Plant pathogen effector and disease resistance gene identification, compositions, and methods of use

ABSTRACT

The compositions and methods are related to plant breeding and methods of identifying and selecting disease resistance genes and plant pathogen effector genes. Provided are methods to identify novel genes that encode plant pathogen effector proteins and proteins providing plant resistance to various diseases and uses thereof. These disease resistant genes are useful in the production of resistant plants through breeding, transgenic modification, or genome editing.

FIELD

The compositions and methods are related to plant breeding and methodsof identifying and selecting disease resistance genes and plant pathogeneffector genes. Provided are methods to identify novel genes that encodeplant pathogen effector proteins and proteins providing plant resistanceto various diseases and uses thereof. These disease resistant genes areuseful in the production of resistant plants through breeding,transgenic modification, or genome editing.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 8052_Seq_List.txt, a creation date of Feb. 23, 2021, and a sizeof 1.629 mb. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

BACKGROUND

Much work has been done on the mechanisms of disease resistance inplants. Some mechanisms of resistance are non-pathogen specific innature, or so-called “non-host resistance.” These may be based on cellwall structure or similar protective mechanisms. However, while plantslack an immune system with circulating antibodies and the otherattributes of a mammalian immune system, they do have other mechanismsto specifically protect against pathogens. The most important and beststudied of these are the plant disease resistance genes, or “R genes.”One of very many reviews of this resistance mechanism and the R genescan be found in Bekhadir et al., (2004), Current Opinion in PlantBiology 7:391-399. There are 5 recognized classes of R genes:intracellular proteins with a nucleotide-binding site (NBS or NB-ARC)and a leucine-rich repeat (LRR); transmembrane proteins with anextracellular LRR domain (TM-LRR); transmembrane and extracellular LRRwith a cytoplasmic kinase domain (TM-CK-LRR); membrane signal anchoredprotein with a coiled-coil cytoplasmic domain (MSAP-CC); and membrane orwall associated kinases with an N-terminal myristylation site (MAK-N orWAK) (See, for example: Cohn, et al., (2001), Immunology, 13:55-62;Dangl, et al. (2001), Nature, 411:826-833). There is a continuous needfor disease-resistant plants and methods to find disease resistantgenes, therefore, there is a need for a faster method of identificationof disease resistance genes with greater throughput.

SUMMARY

Compositions and methods useful in identifying and selecting plantdisease resistance genes, or “R genes,” are provided herein. Thecompositions and methods are useful in selecting resistant plants,creating transgenic resistant plants, and/or creating resistant genomeedited plants. Plants having newly conferred or enhanced resistancevarious plant diseases as compared to control plants are also providedherein.

In some embodiments, a maize or soybean protoplast comprises a predictedpathogen effector gene, a luciferase gene, and a potential maize diseaseresistance gene, wherein the predicted pathogen effector gene ispredicted from a computational analysis. Optionally, the predictedpathogen effector gene and the luciferase reporter gene are bothexpressed from a single expression vector. In another embodiment, aplant comprises a dsRNA targeting a pathogen effector protein, whereinthe pathogen effector protein was identified or validated through aluciferase reporter protoplast assay. In some embodiments, a protoplastcomprises as plant pathogen effector comprising an amino acid sequenceof at least 95% sequence identity, when compared to SEQ ID NOs: 2, 4,10-23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461,463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489,491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545,547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573,575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601,603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629,631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657,659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685,687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713,715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741,743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769,771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797,799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825,827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853,855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881,883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909,911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937,939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965,967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993,995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017,1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041,1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065,1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089,1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113,1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137,1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161,1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185,1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209,1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233,1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257,1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281,1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305,1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329,1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353,1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377,1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401,1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425,1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449,1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473,1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497,1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521,1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545,1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569,1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593,1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617,1619, 1626-1628, or 1631.

In some embodiments, a plant pathogen effector comprises apolynucleotide operably linked to at least one regulatory sequencewherein said polynucleotide comprises a nucleic acid sequence encodingan amino acid sequence of at least 90% or at least 95% sequenceidentity, when compared to SEQ ID NO: 2, 4, 10-23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,275, 277, 279, 281, 283, 285, 289, 291, 293, 295, 297, 299, 301, 303,305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331,333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387,389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415,417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443,445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471,473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499,501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527,529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555,557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583,585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611,613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639,641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667,669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695,697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723,725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751,753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779,781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807,809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835,837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863,865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891,893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919,921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947,949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975,977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003,1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027,1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051,1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075,1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123,1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147,1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171,1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195,1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219,1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243,1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267,1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291,1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315,1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339,1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363,1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387,1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411,1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435,1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459,1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483,1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507,1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531,1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555,1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579,1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603,1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1626-1628, or 1631. Insome embodiments, the polynucleotide encoding SEQ ID NO: 2 or 4comprises a nucleic acid sequence having at least 95% sequence identityto SEQ ID NO: 1 or 3.

In one embodiment, methods for identifying and/or selecting R genes arepresented. In some embodiments, the methods comprise a) identifyingpotential effector proteins from a plant pathogen using computationalanalysis; b) transfecting at least one potential effector gene with aluciferase reporter gene into a maize protoplast; and c) measuringluciferase activity after transfection of the effector and luciferasereporter genes and optionally, transfecting an NLR or other diseaseresistance gene into the protoplast. In some embodiments, the effectorgene and the luciferase reporter gene are both expressed from a singleconstruct. In a further embodiment, the method comprises identifying adisease resistance gene that interacts with the plant pathogen effector.In some aspects, the plant protoplast is derived from a maize plant or asoybean plant. A maize plant or a soybean plant may be susceptible orresistant to the plant pathogen that produces the potential effector.

In some embodiments, a method for identifying a novel mode of action ofa disease resistance gene comprising a) transfecting at least one alleleof a validated plant pathogen effector gene and a luciferase reportergene into a maize protoplast, wherein the maize protoplast is derivedfrom a maize plant resistant to the plant pathogen; b) measuringluciferase activity; and c) selecting maize plants showing no activityin the presence of the effector protein and the luciferase reportergene, wherein the lack of activity indicates the resistant maize plantacts in a different manner than interacting with the plant pathogeneffector protein. In some embodiments the effector gene and theluciferase reporter gene are both expressed from a single construct. Insome embodiments, more than one protoplast are tested which are derivedfrom more than one maize line. In another embodiment, the method furthercomprises identifying a resistance gene or QTL in the selected maizeplant resistant to the plant pathogen.

In some embodiments, a method of validating a causal disease resistancegene comprising a) identifying at least one potential gene in a diseaseresistance loci; b) transfecting a potential resistant gene from thedisease resistance loci, a plant pathogen effector gene, and aluciferase gene into a maize protoplast, wherein the maize protoplast isderived from a maize plant susceptible to the plant pathogen; c)measuring luciferase activity; and d) selecting a gene that produces ahypersensitive response in the presence of the plant pathogen effector,wherein the plant pathogen effector gene has been validated as a plantpathogen effector for the disease correlated with the disease resistanceloci.

In some embodiments, a method of validating a causal disease resistancegene comprising a) identifying at least one potential gene in a diseaseresistance loci in a disease resistance plant; b) transfecting at leastone allele of a plant pathogen effector gene and a luciferase gene intoa maize protoplast, wherein the maize protoplast is derived from thedisease resistance plant; c) measuring luciferase activity; d) selectinga plant that produces luciferase activity in the presence of theeffector gene and luciferase reporter gene; e) transfecting a potentialresistant gene from the disease resistance loci of the disease resistantplant, a plant pathogen effector gene, and a luciferase gene into amaize protoplast, wherein the maize protoplast is derived from a maizeplant susceptible to the plant pathogen; f) measuring luciferaseactivity; and g) selecting a gene that produces a hypersensitiveresponse in the presence of the plant pathogen effector. In someembodiments, the plant pathogen effector gene has been validated as aplant pathogen effector for the disease correlated with the diseaseresistance loci. In another embodiment, disease resistant donor plant isa maize plant or a soybean plant.

In some embodiments, a method of selecting a disease resistant donorplant comprising a) transfecting at least one allele of a plant pathogeneffector gene and a luciferase gene into a maize protoplast, wherein themaize protoplast is derived from a plant resistant to the plantpathogen; b) measuring luciferase activity; and c) selecting a maizeplant that produces a hypersensitive response in the presence of theplant pathogen effector. In some embodiments, the plant pathogeneffector gene has been validated as a plant pathogen effector for thedisease correlated with the disease resistance loci. In anotherembodiment, disease resistant donor plant is a maize plant or a soybeanplant.

In some embodiments, a method to identify homologous plant pathogeneffectors comprising a) transfecting first allele of a plant pathogeneffector gene and a luciferase gene into a maize protoplast; b)measuring luciferase activity; c) selecting a maize plant that producesa hypersensitive response in the presence of the plant pathogeneffector; d) transfecting a second allele of a plant pathogen effectorand a luciferase gene into a selected maize plant derived protoplast;and e) measuring luciferase activity. In some embodiments, the maizeprotoplast is derived from a disease resistant maize plant. In furtherembodiment, the method comprises transfecting at least one moredifferent allele of a plant pathogen effector and a luciferase reportergene into the selected maize protoplast. In some embodiments, the secondallele of a plant pathogen effector was identified from a fieldpopulation of plant pathogens, or the second allele of a plant pathogeneffector indicates the increase in resistance in a field.

In some embodiments, a method of breeding a plant for disease resistancecomprising a) transfecting at least one allele of a plant pathogeneffector gene and a luciferase gene into a maize protoplast, wherein themaize protoplast is derived from at least one maize plant resistant tothe plant pathogen; b) measuring luciferase activity; c) Selecting amaize plant that produces a hypersensitive response in the presence ofthe plant pathogen effector. In further embodiment, the method comprisescrossing the selected maize plant with a second maize plant. In someembodiments, the second allele of a plant pathogen effector wasidentified from a field population of plant pathogens, or the secondallele of a plant pathogen effector indicates the increase in resistancein a field.

In some embodiments, a method to monitor effectiveness of a diseaseresistance gene comprising a) transfecting a plant pathogen effectorgene from a field derived pathogen strain and a luciferase gene into amaize protoplast, wherein the maize protoplast is derived from at leastone maize plant resistant to the plant pathogen; b) measuring luciferaseactivity; c) transfecting a second allele of the plant pathogen effectorgene from the same field derived pathogen strains and a luciferasereporter gene into the maize protoplast; d) measuring luciferaseactivity; and e) comparing luciferase activity from the first and secondplant pathogen effector gene alleles.

In some embodiments, a method to identify non-host resistance genescomprising a) transfecting at least one plant pathogen effector gene anda luciferase gene into a non-host protoplast, wherein the non-hostprotoplast is derived from at least one plant that shows no phenotypicchanges in response to the plant pathogen; b) measuring luciferaseactivity; and c) selecting a non-host plant that has a protoplast thatproduces a hypersensitive response in the presence of the plant pathogeneffector. In further embodiment, the identifying a causal gene for thenon-host resistance to the plant pathogen effector.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overall process flow chart for embodiments of a pathogeneffector—protoplast luciferase assay.

FIG. 2 shows the process for transfection of a plant pathogen effectorand a luciferase reporter gene on one construct into a resistant maizeprotoplast and measuring luciferase activity to determine if theeffector elicits a hypersensitive response in the resistant maizeprotoplast.

FIG. 3 shows a table of bioinformatically identified effectors.

DETAILED DESCRIPTION

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof, and so forth. All technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs unlessclearly indicated otherwise.

For successful colonization or infection of host plants, plant pathogensmust block host defenses or immune responses. The first line of defensein the plant immune system is a basal defense response that is triggeredby pathogen-associated molecular patterns (PAMPs), conserved molecularfeatures among pathogens (e.g. chitin for fungi). PAMP-triggeredimmunity (PTI) involves cell surface pattern recognition receptors(PRRs). Pathogens secrete effectors, which are generally small uniqueprotein with no known functions, to modulate host cell physiology,suppress PTI and promote susceptibility. In turn, plants have developeda second line of defense, effector-triggered immunity (ETI), whichinvolves the detection of specific avirulence effectors by intracellularreceptors. These intracellular immune receptors are nucleotide-bindingdomain and leucine-rich repeat (NLR) proteins. NLRs recognize theircognate effectors either through direct interaction or through indirectdetection. This recognition usually triggers the hypersensitive response(HR), a programmed cell death and the hallmark of ETI.

The NBS-LRR (“NLR”) group of R-genes is the largest class of R-genesdiscovered to date. In Arabidopsis thaliana, over 150 are predicted tobe present in the genome (Meyers, et al., (2003), Plant Cell,15:809-834; Monosi, et al., (2004), Theoretical and Applied Genetics,109:1434-1447), while in rice, approximately 500 NLR genes have beenpredicted (Monosi, (2004) supra). The NBS-LRR class of R genes iscomprised of two subclasses. Class 1 NLR genes contain aTIR-Toll/Interleukin-1 like domain at their N′ terminus; which to datehave only been found in dicots (Meyers, (2003) supra; Monosi, (2004)supra). The second class of NBS-LRR contain either a coiled-coil domainor an (nt) domain at their N terminus (Bai, et al. (2002) GenomeResearch, 12:1871-1884; Monosi, (2004) supra; Pan, et al., (2000),Journal of Molecular Evolution, 50:203-213). Class 2 NBS-LRR have beenfound in both dicot and monocot species. (Bai, (2002) supra; Meyers,(2003) supra; Monosi, (2004) supra; Pan, (2000) supra).

The NBS domain of the gene appears to have a role in signaling in plantdefense mechanisms (van der Biezen, et al., (1998), Current Biology: CB,8:R226-R227). The LRR region appears to be the region that interactswith the pathogen AVR products (Michelmore, et al., (1998), Genome Res.,8:1113-1130; Meyers, (2003) supra). This LRR region in comparison withthe NB-ARC (NBS) domain is under a much greater selection pressure todiversify (Michelmore, (1998) supra; Meyers, (2003) supra; Palomino, etal., (2002), Genome Research, 12:1305-1315). LRR domains are found inother contexts as well; these 20-29-residue motifs are present in tandemarrays in a number of proteins with diverse functions, such ashormone—receptor interactions, enzyme inhibition, cell adhesion andcellular trafficking. A number of recent studies revealed theinvolvement of LRR proteins in early mammalian development, neuraldevelopment, cell polarization, regulation of gene expression andapoptosis signaling.

A resistance gene of the embodiments of the present disclosure encodes anovel R gene. The most numerous R genes correspond to the NBS-LRR type.There have also been many identified WAK type R genes. While multipleNBS-LRR genes have been described, they may differ widely in theirresponse to different pathogens and exact action.

Positional cloning (or map-based cloning) has been the major method inidentifying causal genes responsible for variations in diseaseresistance. In this approach, a resistance line is crossed to asusceptible line to generate a mapping population segregating forresistance and susceptibility. Linkage mapping is performed withgenotyping and phenotyping data to detect disease QTL (QuantitativeTrait Loci), or a disease resistance loci. A major disease QTL is“mendenlized” through back-crossing to the susceptible parents andvalidated. A validated QTL is then fine mapped into a small intervalwith a large segregating population (typically with over 3000individuals). Sequences covering the QTL interval are obtained from theresistance line via BAC clone identification/sequencing or genomesequencing. The genome sequence is annotated, candidate genes identifiedand tested in transgenic plants. The candidate gene conferringresistance in transgenic plants is the causal gene underlying thedisease QTL.

As used to herein, “disease resistant” or “have resistance to a disease”refers to a plant showing increase resistance to a disease compared to acontrol plant, which is a susceptible plant. Disease resistance maymanifest in fewer and/or smaller lesions, increased plant health,increased yield, increased root mass, increased plant vigor, less or nodiscoloration, increased growth, reduced necrotic area, or reducedwilting.

Disease affecting maize plants include, but are not limited to,bacterial leaf blight and stalk rot; bacterial leaf spot; bacterialstripe; chocolate spot; goss's bacterial wilt and blight; holcus spot;purple leaf sheath; seed rot-seedling blight; bacterial wilt; cornstunt; anthracnose leaf blight; anthracnose stalk rot; aspergillus earand kernel rot; banded leaf and sheath spot; black bundle disease; blackkernel rot; borde blanco; brown spot; black spot; stalk rot;cephalosporium kernel rot; charcoal rot; corticium ear rot; curvularialeaf spot; didymella leaf spot; diplodia ear rot and stalk rot; diplodiaear rot; seed rot; corn seedling blight; diplodia leaf spot or leafstreak; downy mildews; brown stripe downy mildew; crazy top downymildew; green ear downy mildew; graminicola downy mildew; java downymildew; philippine downy mildew; sorghum downy mildew; spontaneum downymildew; sugarcane downy mildew; dry ear rot; ergot; horse's tooth; corneyespot; fusarium ear and stalk rot; fusarium blight; seedling root rot;gibberella ear and stalk rot; gray ear rot; gray leaf spot; cercosporaleaf spot; helminthosporium root rot; hormodendrum ear rot; cladosporiumrot; hyalothyridium leaf spot; late wilt; northern leaf blight; whiteblast; crown stalk rot; corn stripe; northern leaf spot;helminthosporium ear rot; penicillium ear rot; corn blue eye; blue mold;phaeocytostroma stalk rot and root rot; phaeosphaeria leaf spot;physalospora ear rot; botryosphaeria ear rot; pyrenochaeta stalk rot androot rot; pythium root rot; pythium stalk rot; red kernel disease;rhizoctonia ear rot; sclerotial rot; rhizoctonia root rot and stalk rot;rostratum leaf spot; common corn rust; southern corn rust; tropical cornrust; sclerotium ear rot; southern blight; selenophoma leaf spot; sheathrot; shuck rot; silage mold; common smut; false smut; head smut;southern corn leaf blight and stalk rot; southern leaf spot; tar spot;trichoderma ear rot and root rot; white ear rot, root and stalk rot;yellow leaf blight; zonate leaf spot; american wheat striate (wheatstriate mosaic); barley stripe mosaic; barley yellow dwarf; bromemosaic; cereal chlorotic mottle; lethal necrosis (maize lethal necrosisdisease); cucumber mosaic; johnsongrass mosaic; maize bushy stunt; maizechlorotic dwarf; maize chlorotic mottle; maize dwarf mosaic; maize leaffleck; maize pellucid ringspot; maize rayado fino; maize red leaf andred stripe; maize red stripe; maize ring mottle; maize rough dwarf;maize sterile stunt; maize streak; maize stripe; maize tassel abortion;maize vein enation; maize wallaby ear; maize white leaf; maize whiteline mosaic; millet red leaf; and northern cereal mosaic.

Disease affecting rice plants include, but are not limited to, bacterialblight; bacterial leaf streak; foot rot; grain rot; sheath brown rot;blast; brown spot; crown sheath rot; downy mildew; eyespot; false smut;kernel smut; leaf smut; leaf scald; narrow brown leaf spot; root rot;seedling blight; sheath blight; sheath rot; sheath spot; alternaria leafspot; and stem rot.

Disease affecting soybean plants include, but are not limited to,alternaria leaf spot; anthracnose; black leaf blight; black root rot;brown spot; brown stem rot; charcoal rot; choanephora leaf blight; downymildew; drechslera blight; frogeye leaf spot; leptosphaerulina leafspot; mycoleptodiscus root rot; neocosmospora stem rot; phomopsis seeddecay; phytophthora root and stem rot; phyllosticta leaf spot;phymatotrichum root rot; pod and stem blight; powdery mildew; purpleseed stain; pyrenochaeta leaf spot; pythium rot; red crown rot;dactuliophora leaf spot; rhizoctonia aerial blight; rhizoctonia root andstem rot; rust; scab; sclerotinia stem rot; sclerotium blight; stemcanker; stemphylium leaf blight; sudden death syndrome; target spot;yeast spot; lance nematode; lesion nematode; pin nematode; reniformnematode; ring nematode; root-knot nematode; sheath nematode; cystnematode; spiral nematode; sting nematode; stubby root nematode; stuntnematode; alfalfa mosaic; bean pod mottle; bean yellow mosaic; brazilianbud blight; chlorotic mottle; yellow mosaic; peanut mottle; peanutstripe; peanut stunt; chlorotic mottle; crinkle leaf; dwarf; severestunt; and tobacco ringspot or bud blight.

Disease affecting canola plants include, but are not limited to,bacterial black rot; bacterial leaf spot; bacterial pod rot; bacterialsoft rot; scab; crown gall; alternaria black spot; anthracnose; blackleg; black mold rot; black root; brown girdling root rot; cercosporaleaf spot; clubroot; downy mildew; fusarium wilt; gray mold; head rot;leaf spot; light leaf spot; pod rot; powdery mildew; ring spot; rootrot; sclerotinia stem rot; seed rot, damping-off; root gall smut;southern blight; verticillium wilt; white blight; white leaf spot;staghead; yellows; crinkle virus; mosaic virus; yellows virus;

Disease affecting sunflower plants include, but are not limited to,apical chlorosis; bacterial leaf spot; bacterial wilt; crown gall;erwinia stalk rot and head rot; lternaria leaf blight, stem spot andhead rot; botrytis head rot; charcoal rot; downy mildew; fusarium stalkrot; fusarium wilt; myrothecium leaf and stem spot; phialophora yellows;phoma black stem; phomopsis brown stem canker; phymatotrichum root rot;phytophthora stem rot; powdery mildew; pythium seedling blight and rootrot; rhizoctonia seedling blight; rhizopus head rot; sunflower rust;sclerotium basal stalk and root rot; septoria leaf spot; verticilliumwilt; white rust; yellow rust; dagger; pin; lesion; reniform; root knot;and chlorotic mottle;

Disease affecting sorghum plants include, but are not limited to,bacterial leaf spot; bacterial leaf streak; bacterial leaf stripe;acremonium wilt; anthracnose; charcoal rot; crazy top downy mildew;damping-off and seed rot; ergot; fusarium head blight, root and stalkrot; grain storage mold; gray leaf spot; latter leaf spot; leaf blight;milo disease; oval leaf spot; pokkah boeng; pythium root rot; rough leafspot; rust; seedling blight and seed rot; smut, covered kernel; smut,head; smut, loose kernel; sooty stripe; downy mildew; tar spot; targetleaf spot; and zonate leaf spot and sheath blight.

A plant having disease resistance may have 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increasedresistance to a disease compared to a control plant. In someembodiments, a plant may have 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100% increased plant health in thepresence of a disease compared to a control plant

As used herein, the term “clustering” or “clustering approach” meanspooling and clustering sequences in a location-agnostic manner using anearest neighbor joining algorithm, hierarchical clustering such asWard's method, a maximum likelihood method, or any other clusteringalgorithm or method.

The term “crossed” or “cross” refers to a sexual cross and involved thefusion of two haploid gametes via pollination to produce diploid progeny(e.g., cells, seeds or plants). The term encompasses both thepollination of one plant by another and selfing (or self-pollination,e.g., when the pollen and ovule are from the same plant).

An “elite line” is any line that has resulted from breeding andselection for superior agronomic performance.

An “exotic strain,” a “tropical line,” or an “exotic germplasm” is astrain derived from a plant not belonging to an available elite line orstrain of germplasm. In the context of a cross between two plants orstrains of germplasm, an exotic germplasm is not closely related bydescent to the elite germplasm with which it is crossed. Most commonly,the exotic germplasm is not derived from any known elite line, butrather is selected to introduce novel genetic elements (typically novelalleles) into a breeding program.

A “favorable allele” is the allele at a particular locus (a marker, aQTL, a gene etc.) that confers, or contributes to, an agronomicallydesirable phenotype, e.g., disease resistance, and that allows theidentification of plants with that agronomically desirable phenotype. Afavorable allele of a marker is a marker allele that segregates with thefavorable phenotype.

“Genetic markers” are nucleic acids that are polymorphic in a populationand where the alleles of which can be detected and distinguished by oneor more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and thelike. The term also refers to nucleic acid sequences complementary tothe genomic sequences, such as nucleic acids used as probes. Markerscorresponding to genetic polymorphisms between members of a populationcan be detected by methods well-established in the art. These include,e.g., PCR-based sequence specific amplification methods, detection ofrestriction fragment length polymorphisms (RFLP), detection of isozymemarkers, detection of polynucleotide polymorphisms by allele specifichybridization (ASH), detection of amplified variable sequences of theplant genome, detection of self-sustained sequence replication,detection of simple sequence repeats (SSRs), detection of singlenucleotide polymorphisms (SNPs), or detection of amplified fragmentlength polymorphisms (AFLPs). Well established methods are also knownfor the detection of expressed sequence tags (ESTs) and SSR markersderived from EST sequences and randomly amplified polymorphic DNA(RAPD).

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture, or moregenerally, all individuals within a species or for several species(e.g., maize germplasm collection or Andean germplasm collection). Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells, that can be cultured into a whole plant.

A “haplotype” is the genotype of an individual at a plurality of geneticloci, i.e. a combination of alleles. Typically, the genetic locidescribed by a haplotype are physically and genetically linked, i.e., onthe same chromosome segment.

The term “heterogeneity” is used to indicate that individuals within thegroup differ in genotype at one or more specific loci.

The heterotic response of material, or “heterosis”, can be defined byperformance which exceeds the average of the parents (or high parent)when crossed to other dissimilar or unrelated groups.

A “heterotic group” comprises a set of genotypes that perform well whencrossed with genotypes from a different heterotic group (Hallauer et al.(1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley(ed.) Corn and corn improvement). Inbred lines are classified intoheterotic groups, and are further subdivided into families within aheterotic group, based on several criteria such as pedigree, molecularmarker-based associations, and performance in hybrid combinations (Smithet al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely usedheterotic groups in the United States are referred to as “Iowa StiffStalk Synthetic” (also referred to herein as “stiff stalk”) and“Lancaster” or “Lancaster Sure Crop” (sometimes referred to as NSS, ornon-Stiff Stalk).

Some heterotic groups possess the traits needed to be a female parent,and others, traits for a male parent. For example, in maize, yieldresults from public inbreds released from a population called BSSS (IowaStiff Stalk Synthetic population) has resulted in these inbreds andtheir derivatives becoming the female pool in the central Corn Belt.BSSS inbreds have been crossed with other inbreds, e.g. SD 105 and MaizAmargo, and this general group of materials has become known as StiffStalk Synthetics (SSS) even though not all of the inbreds are derivedfrom the original BSSS population (Mikel and Dudley (2006) Crop Sci:46:1193-1205). By default, all other inbreds that combine well with theSSS inbreds have been assigned to the male pool, which for lack of abetter name has been designated as NSS, i.e. Non-Stiff Stalk. This groupincludes several major heterotic groups such as Lancaster Surecrop,lodent, and Leaming Corn.

The term “homogeneity” indicates that members of a group have the samegenotype at one or more specific loci.

The term “hybrid” refers to the progeny obtained between the crossing ofat least two genetically dissimilar parents.

The term “inbred” refers to a line that has been bred for genetichomogeneity.

The term “indel” refers to an insertion or deletion, wherein one linemay be referred to as having an inserted nucleotide or piece of DNArelative to a second line, or the second line may be referred to ashaving a deleted nucleotide or piece of DNA relative to the first line.

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny via a sexual cross between twoparents of the same species, where at least one of the parents has thedesired allele in its genome. Alternatively, for example, transmissionof an allele can occur by recombination between two donor genomes, e.g.,in a fused protoplast, where at least one of the donor protoplasts hasthe desired allele in its genome. The desired allele can be, e.g.,detected by a marker that is associated with a phenotype, at a QTL, atransgene, or the like. In any case, offspring comprising the desiredallele can be repeatedly backcrossed to a line having a desired geneticbackground and selected for the desired allele, to result in the allelebecoming fixed in a selected genetic background.

The process of “introgressing” is often referred to as “backcrossing”when the process is repeated two or more times.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci (isogenic or near isogenic). A“subline” refers to an inbred subset of descendants that are geneticallydistinct from other similarly inbred subsets descended from the sameprogenitor.

As used herein, the term “linkage” is used to describe the degree withwhich one marker locus is associated with another marker locus or someother locus. The linkage relationship between a molecular marker and alocus affecting a phenotype is given as a “probability” or “adjustedprobability”. Linkage can be expressed as a desired limit or range. Forexample, in some embodiments, any marker is linked (genetically andphysically) to any other marker when the markers are separated by lessthan 50, 40, 30, 25, 20, or 15 map units (or cM) of a single meiosis map(a genetic map based on a population that has undergone one round ofmeiosis, such as e.g. an F₂; the IBM2 maps consist of multiple meiosis).In some aspects, it is advantageous to define a bracketed range oflinkage, for example, between 10 and 20 cM, between 10 and 30 cM, orbetween 10 and 40 cM. The more closely a marker is linked to a secondlocus, the better an indicator for the second locus that marker becomes.Thus, “closely linked loci” such as a marker locus and a second locusdisplay an inter-locus recombination frequency of 10% or less,preferably about 9% or less, still more preferably about 8% or less, yetmore preferably about 7% or less, still more preferably about 6% orless, yet more preferably about 5% or less, still more preferably about4% or less, yet more preferably about 3% or less, and still morepreferably about 2% or less. In highly preferred embodiments, therelevant loci display a recombination frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Two loci that are localized to thesame chromosome, and at such a distance that recombination between thetwo loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be“in proximity to” each other. Since one cM is the distance between twomarkers that show a 1% recombination frequency, any marker is closelylinked (genetically and physically) to any other marker that is in closeproximity, e.g., at or less than 10 cM distant. Two closely linkedmarkers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2,1, 0.75, 0.5 or 0.25 cM or less from each other.

The term “linkage disequilibrium” refers to a non-random segregation ofgenetic loci or traits (or both). In either case, linkage disequilibriumimplies that the relevant loci are within sufficient physical proximityalong a length of a chromosome so that they segregate together withgreater than random (i.e., non-random) frequency. Markers that showlinkage disequilibrium are considered linked. Linked loci co-segregatemore than 50% of the time, e.g., from about 51% to about 100% of thetime. In other words, two markers that co-segregate have a recombinationfrequency of less than 50% (and by definition, are separated by lessthan 50 cM on the same linkage group.) As used herein, linkage can bebetween two markers, or alternatively between a marker and a locusaffecting a phenotype. A marker locus can be “associated with” (linkedto) a trait. The degree of linkage of a marker locus and a locusaffecting a phenotypic trait is measured, e.g., as a statisticalprobability of co-segregation of that molecular marker with thephenotype (e.g., an F statistic or LOD score).

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill, W. G. andRobertson, A, Theor. Appl. Genet. 38:226-231(1968). When r²=1, completeLD exists between the two marker loci, meaning that the markers have notbeen separated by recombination and have the same allele frequency. Ther² value will be dependent on the population used. Values for r² above ⅓indicate sufficiently strong LD to be useful for mapping (Ardlie et al.,Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are in linkagedisequilibrium when r² values between pairwise marker loci are greaterthan or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

As used herein, “linkage equilibrium” describes a situation where twomarkers independently segregate, i.e., sort among progeny randomly.Markers that show linkage equilibrium are considered unlinked (whetheror not they lie on the same chromosome).

A “locus” is a position on a chromosome, e.g. where a nucleotide, gene,sequence, or marker is located.

The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science255:803-804 (1992)) is used in genetic interval mapping to describe thedegree of linkage between two marker loci. A LOD score of three betweentwo markers indicates that linkage is 1000 times more likely than nolinkage, while a LOD score of two indicates that linkage is 100 timesmore likely than no linkage. LOD scores greater than or equal to two maybe used to detect linkage. LOD scores can also be used to show thestrength of association between marker loci and quantitative traits in“quantitative trait loci” mapping. In this case, the LOD score's size isdependent on the closeness of the marker locus to the locus affectingthe quantitative trait, as well as the size of the quantitative traiteffect.

The term “plant” includes whole plants, plant cells, plant protoplast,plant cell or tissue culture from which plants can be regenerated, plantcalli, plant clumps and plant cells that are intact in plants or partsof plants, such as seeds, flowers, cotyledons, leaves, stems, buds,roots, root tips and the like. As used herein, a “modified plant” meansany plant that has a genetic change due to human intervention. Amodified plant may have genetic changes introduced through planttransformation, genome editing, or conventional plant breeding

A “marker” is a means of finding a position on a genetic or physicalmap, or else linkages among markers and trait loci (loci affectingtraits). The position that the marker detects may be known via detectionof polymorphic alleles and their genetic mapping, or else byhybridization, sequence match or amplification of a sequence that hasbeen physically mapped. A marker can be a DNA marker (detects DNApolymorphisms), a protein (detects variation at an encoded polypeptide),or a simply inherited phenotype (such as the ‘waxy’ phenotype). A DNAmarker can be developed from genomic nucleotide sequence or fromexpressed nucleotide sequences (e.g., from a spliced RNA or a cDNA).Depending on the DNA marker technology, the marker will consist ofcomplementary primers flanking the locus and/or complementary probesthat hybridize to polymorphic alleles at the locus. A DNA marker, or agenetic marker, can also be used to describe the gene, DNA sequence ornucleotide on the chromosome itself (rather than the components used todetect the gene or DNA sequence) and is often used when that DNA markeris associated with a particular trait in human genetics (e.g. a markerfor breast cancer). The term marker locus is the locus (gene, sequenceor nucleotide) that the marker detects.

Markers that detect genetic polymorphisms between members of apopulation are well-established in the art. Markers can be defined bythe type of polymorphism that they detect and also the marker technologyused to detect the polymorphism. Marker types include but are notlimited to, e.g., detection of restriction fragment length polymorphisms(RFLP), detection of isozyme markers, randomly amplified polymorphic DNA(RAPD), amplified fragment length polymorphisms (AFLPs), detection ofsimple sequence repeats (SSRs), detection of amplified variablesequences of the plant genome, detection of self-sustained sequencereplication, or detection of single nucleotide polymorphisms (SNPs).SNPs can be detected e.g. via DNA sequencing, PCR-based sequencespecific amplification methods, detection of polynucleotidepolymorphisms by allele specific hybridization (ASH), dynamicallele-specific hybridization (DASH), molecular beacons, microarrayhybridization, oligonucleotide ligase assays, Flap endonucleases, 5′endonucleases, primer extension, single strand conformation polymorphism(SSCP) or temperature gradient gel electrophoresis (TGGE). DNAsequencing, such as the pyrosequencing technology has the advantage ofbeing able to detect a series of linked SNP alleles that constitute ahaplotype. Haplotypes tend to be more informative (detect a higher levelof polymorphism) than SNPs.

A “marker allele”, alternatively an “allele of a marker locus”, canrefer to one of a plurality of polymorphic nucleotide sequences found ata marker locus in a population.

“Marker assisted selection” (of MAS) is a process by which individualplants are selected based on marker genotypes.

“Marker assisted counter-selection” is a process by which markergenotypes are used to identify plants that will not be selected,allowing them to be removed from a breeding program or planting.

A “marker haplotype” refers to a combination of alleles at a markerlocus.

A “marker locus” is a specific chromosome location in the genome of aspecies where a specific marker can be found. A marker locus can be usedto track the presence of a second linked locus, e.g., one that affectsthe expression of a phenotypic trait. For example, a marker locus can beused to monitor segregation of alleles at a genetically or physicallylinked locus.

The term “molecular marker” may be used to refer to a genetic marker, asdefined above, or an encoded product thereof (e.g., a protein) used as apoint of reference when identifying a linked locus. A marker can bederived from genomic nucleotide sequences or from expressed nucleotidesequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encodedpolypeptide. The term also refers to nucleic acid sequencescomplementary to or flanking the marker sequences, such as nucleic acidsused as probes or primer pairs capable of amplifying the markersequence. A “molecular marker probe” is a nucleic acid sequence ormolecule that can be used to identify the presence of a marker locus,e.g., a nucleic acid probe that is complementary to a marker locussequence. Alternatively, in some aspects, a marker probe refers to aprobe of any type that is able to distinguish (i.e., genotype) theparticular allele that is present at a marker locus. Nucleic acids are“complementary” when they specifically hybridize in solution. Some ofthe markers described herein are also referred to as hybridizationmarkers when located on an indel region, such as the non-collinearregion described herein. This is because the insertion region is, bydefinition, a polymorphism vis a vis a plant without the insertion.Thus, the marker need only indicate whether the indel region is presentor absent. Any suitable marker detection technology may be used toidentify such a hybridization marker, e.g. SNP technology is used in theexamples provided herein.

An allele “negatively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that a desired trait ortrait form will not occur in a plant comprising the allele.

The term “phenotype”, “phenotypic trait”, or “trait” can refer to theobservable expression of a gene or series of genes. The phenotype can beobservable to the naked eye, or by any other means of evaluation knownin the art, e.g., weighing, counting, measuring (length, width, angles,etc.), microscopy, biochemical analysis, or an electromechanical assay.In some cases, a phenotype is directly controlled by a single gene orgenetic locus, i.e., a “single gene trait” or a “simply inheritedtrait”. In the absence of large levels of environmental variation,single gene traits can segregate in a population to give a “qualitative”or “discrete” distribution, i.e. the phenotype falls into discreteclasses. In other cases, a phenotype is the result of several genes andcan be considered a “multigenic trait” or a “complex trait”. Multigenictraits segregate in a population to give a “quantitative” or“continuous” distribution, i.e. the phenotype cannot be separated intodiscrete classes. Both single gene and multigenic traits can be affectedby the environment in which they are being expressed, but multigenictraits tend to have a larger environmental component.

A “physical map” of the genome is a map showing the linear order ofidentifiable landmarks (including genes, markers, etc.) on chromosomeDNA. However, in contrast to genetic maps, the distances betweenlandmarks are absolute (for example, measured in base pairs or isolatedand overlapping contiguous genetic fragments) and not based on geneticrecombination (that can vary in different populations).

A “polymorphism” is a variation in the DNA between two or moreindividuals within a population. A polymorphism preferably has afrequency of at least 1% in a population. A useful polymorphism caninclude a single nucleotide polymorphism (SNP), a simple sequence repeat(SSR), or an insertion/deletion polymorphism, also referred to herein asan “indel”.

A “production marker” or “production SNP marker” is a marker that hasbeen developed for high-throughput purposes. Production SNP markers aredeveloped to detect specific polymorphisms and are designed for use witha variety of chemistries and platforms.

The term “quantitative trait locus” or “QTL” refers to a region of DNAthat is associated with the differential expression of a quantitativephenotypic trait in at least one genetic background, e.g., in at leastone breeding population. The region of the QTL encompasses or is closelylinked to the gene or genes that affect the trait in question.

A “reference sequence” or a “consensus sequence” is a defined sequenceused as a basis for sequence comparison. The reference sequence for amarker is obtained by sequencing a number of lines at the locus,aligning the nucleotide sequences in a sequence alignment program (e.g.Sequencher), and then obtaining the most common nucleotide sequence ofthe alignment. Polymorphisms found among the individual sequences areannotated within the consensus sequence. A reference sequence is notusually an exact copy of any individual DNA sequence, but represents anamalgam of available sequences and is useful for designing primers andprobes to polymorphisms within the sequence.

An “unfavorable allele” of a marker is a marker allele that segregateswith the unfavorable plant phenotype, therefore providing the benefit ofidentifying plants that can be removed from a breeding program orplanting.

The term “yield” refers to the productivity per unit area of aparticular plant product of commercial value. Yield is affected by bothgenetic and environmental factors. “Agronomics”, “agronomic traits”, and“agronomic performance” refer to the traits (and underlying geneticelements) of a given plant variety that contribute to yield over thecourse of growing season. Individual agronomic traits include emergencevigor, vegetative vigor, stress tolerance, disease resistance ortolerance, herbicide resistance, branching, flowering, seed set, seedsize, seed density, standability, threshability and the like. Yield is,therefore, the final culmination of all agronomic traits.

Marker loci that demonstrate statistically significant co-segregationwith a disease resistance trait that confers broad resistance against aspecified disease or diseases are provided herein. Detection of theseloci or additional linked loci and the resistance gene may be used inmarker assisted selection as part of a breeding program to produceplants that have resistance to a disease or diseases.

Genetic Mapping

It has been recognized for quite some time that specific genetic locicorrelating with particular phenotypes, such as disease resistance, canbe mapped in an organism's genome. The plant breeder can advantageouslyuse molecular markers to identify desired individuals by detectingmarker alleles that show a statistically significant probability ofco-segregation with a desired phenotype, manifested as linkagedisequilibrium. By identifying a molecular marker or clusters ofmolecular markers that co-segregate with a trait of interest, thebreeder is able to rapidly select a desired phenotype by selecting forthe proper molecular marker allele (a process called marker-assistedselection, or MAS).

A variety of methods well known in the art are available for detectingmolecular markers or clusters of molecular markers that co-segregatewith a trait of interest, such as a disease resistance trait. The basicidea underlying these methods is the detection of markers, for whichalternative genotypes (or alleles) have significantly different averagephenotypes. Thus, one makes a comparison among marker loci of themagnitude of difference among alternative genotypes (or alleles) or thelevel of significance of that difference. Trait genes are inferred to belocated nearest the marker(s) that have the greatest associatedgenotypic difference. Two such methods used to detect trait loci ofinterest are: 1) Population-based association analysis (i.e. associationmapping) and 2) Traditional linkage analysis.

Association Mapping

Understanding the extent and patterns of linkage disequilibrium (LD) inthe genome is a prerequisite for developing efficient associationapproaches to identify and map quantitative trait loci (QTL). Linkagedisequilibrium (LD) refers to the non-random association of alleles in acollection of individuals. When LD is observed among alleles at linkedloci, it is measured as LD decay across a specific region of achromosome. The extent of the LD is a reflection of the recombinationalhistory of that region. The average rate of LD decay in a genome canhelp predict the number and density of markers that are required toundertake a genome-wide association study and provides an estimate ofthe resolution that can be expected.

Association or LD mapping aims to identify significantgenotype-phenotype associations. It has been exploited as a powerfultool for fine mapping in outcrossing species such as humans (Corder etal. (1994) “Protective effect of apolipoprotein-E type-2 allele forlate-onset Alzheimer-disease,” Nat Genet 7:180-184; Hastbacka et al.(1992) “Linkage disequilibrium mapping in isolated founder populations:diastrophic dysplasia in Finland,” Nat Genet 2:204-211; Kerem et al.(1989) “Identification of the cystic fibrosis gene: genetic analysis,”Science 245:1073-1080) and maize (Remington et al., (2001) “Structure oflinkage disequilibrium and phenotype associations in the maizegenome,”Proc Natl Acad Sci USA 98:11479-11484; Thornsberry et al. (2001)“Dwarf8 polymorphisms associate with variation in flowering time,” NatGenet 28:286-289; reviewed by Flint-Garcia et al. (2003) “Structure oflinkage disequilibrium in plants,” Annu Rev Plant Biol. 54:357-374),where recombination among heterozygotes is frequent and results in arapid decay of LD. In inbreeding species where recombination amonghomozygous genotypes is not genetically detectable, the extent of LD isgreater (i.e., larger blocks of linked markers are inherited together)and this dramatically enhances the detection power of associationmapping (Wall and Pritchard (2003) “Haplotype blocks and linkagedisequilibrium in the human genome,” Nat Rev Genet 4:587-597).

The recombinational and mutational history of a population is a functionof the mating habit as well as the effective size and age of apopulation. Large population sizes offer enhanced possibilities fordetecting recombination, while older populations are generallyassociated with higher levels of polymorphism, both of which contributeto observably accelerated rates of LD decay. On the other hand, smallereffective population sizes, e.g., those that have experienced a recentgenetic bottleneck, tend to show a slower rate of LD decay, resulting inmore extensive haplotype conservation (Flint-Garcia et al. (2003)“Structure of linkage disequilibrium in plants,” Annu Rev Plant Biol.54:357-374).

Elite breeding lines provide a valuable starting point for associationanalyses. Association analyses use quantitative phenotypic scores (e.g.,disease tolerance rated from one to nine for each line) in the analysis(as opposed to looking only at tolerant versus resistant allelefrequency distributions in intergroup allele distribution types ofanalysis). The availability of detailed phenotypic performance datacollected by breeding programs over multiple years and environments fora large number of elite lines provides a valuable dataset for geneticmarker association mapping analyses. This paves the way for a seamlessintegration between research and application and takes advantage ofhistorically accumulated data sets. However, an understanding of therelationship between polymorphism and recombination is useful indeveloping appropriate strategies for efficiently extracting maximuminformation from these resources.

This type of association analysis neither generates nor requires any mapdata, but rather is independent of map position. This analysis comparesthe plants' phenotypic score with the genotypes at the various loci.Subsequently, any suitable map (for example, a composite map) canoptionally be used to help observe distribution of the identified QTLmarkers and/or QTL marker clustering using previously determined maplocations of the markers.

Traditional Linkage Analysis

The same principles underlie traditional linkage analysis; however, LDis generated by creating a population from a small number of founders.The founders are selected to maximize the level of polymorphism withinthe constructed population, and polymorphic sites are assessed for theirlevel of cosegregation with a given phenotype. A number of statisticalmethods have been used to identify significant marker-traitassociations. One such method is an interval mapping approach (Landerand Botstein, Genetics 121:185-199 (1989), in which each of manypositions along a genetic map (say at 1 cM intervals) is tested for thelikelihood that a gene controlling a trait of interest is located atthat position. The genotype/phenotype data are used to calculate foreach test position a LOD score (log of likelihood ratio). When the LODscore exceeds a threshold value, there is significant evidence for thelocation of a gene controlling the trait of interest at that position onthe genetic map (which will fall between two particular marker loci).

Marker loci that demonstrate statistically significant co-segregationwith a disease resistance trait, as determined by traditional linkageanalysis and by whole genome association analysis, are provided herein.Detection of these loci or additional linked loci can be used in markerassisted breeding programs to produce plants having disease resistance.

Activities in marker assisted breeding programs may include but are notlimited to: selecting among new breeding populations to identify whichpopulation has the highest frequency of favorable nucleic acid sequencesbased on historical genotype and agronomic trait associations, selectingfavorable nucleic acid sequences among progeny in breeding populations,selecting among parental lines based on prediction of progenyperformance, and advancing lines in germplasm improvement activitiesbased on presence of favorable nucleic acid sequences.

Markers and Linkage Relationships

A common measure of linkage is the frequency with which traitscosegregate. This can be expressed as a percentage of cosegregation(recombination frequency) or in centiMorgans (cM). The cM is a unit ofmeasure of genetic recombination frequency. One cM is equal to a 1%chance that a trait at one genetic locus will be separated from a traitat another locus due to crossing over in a single generation (meaningthe traits segregate together 99% of the time). Because chromosomaldistance is approximately proportional to the frequency of crossing overevents between traits, there is an approximate physical distance thatcorrelates with recombination frequency.

Marker loci are themselves traits and can be assessed according tostandard linkage analysis by tracking the marker loci duringsegregation. Thus, one cM is equal to a 1% chance that a marker locuswill be separated from another locus, due to crossing over in a singlegeneration.

The closer a marker is to a gene controlling a trait of interest, themore effective and advantageous that marker is as an indicator for thedesired trait. Closely linked loci display an inter-locus cross-overfrequency of about 10% or less, preferably about 9% or less, still morepreferably about 8% or less, yet more preferably about 7% or less, stillmore preferably about 6% or less, yet more preferably about 5% or less,still more preferably about 4% or less, yet more preferably about 3% orless, and still more preferably about 2% or less. In highly preferredembodiments, the relevant loci (e.g., a marker locus and a target locus)display a recombination frequency of about 1% or less, e.g., about 0.75%or less, more preferably about 0.5% or less, or yet more preferablyabout 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or lessapart. Put another way, two loci that are localized to the samechromosome, and at such a distance that recombination between the twoloci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be“proximal to” each other.

Although particular marker alleles can co-segregate with the diseaseresistance trait, it is important to note that the marker locus is notnecessarily responsible for the expression of the disease resistancephenotype. For example, it is not a requirement that the markerpolynucleotide sequence be part of a gene that is responsible for thedisease resistant phenotype (for example, is part of the gene openreading frame). The association between a specific marker allele and thedisease resistance trait is due to the original “coupling” linkage phasebetween the marker allele and the allele in the ancestral line fromwhich the allele originated. Eventually, with repeated recombination,crossing over events between the marker and genetic locus can changethis orientation. For this reason, the favorable marker allele maychange depending on the linkage phase that exists within the parenthaving resistance to the disease that is used to create segregatingpopulations. This does not change the fact that the marker can be usedto monitor segregation of the phenotype. It only changes which markerallele is considered favorable in a given segregating population.

Methods presented herein include detecting the presence of one or moremarker alleles associated with disease resistance in a plant and thenidentifying and/or selecting plants that have favorable alleles at thosemarker loci. Markers have been identified herein as being associatedwith the disease resistance trait and hence can be used to predictdisease resistance in a plant. Any marker within 50 cM, 40 cM, 30 cM, 20cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM,0.75 cM, 0.5 cM or 0.25 cM (based on a single meiosis based genetic map)could also be used to predict disease resistance in a plant.

Marker Assisted Selection

Molecular markers can be used in a variety of plant breedingapplications (e.g. see Staub et al. (1996) Hortscience 31: 729-741;Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of themain areas of interest is to increase the efficiency of backcrossing andintrogressing genes using marker-assisted selection (MAS). A molecularmarker that demonstrates linkage with a locus affecting a desiredphenotypic trait provides a useful tool for the selection of the traitin a plant population. This is particularly true where the phenotype ishard to assay. Since DNA marker assays are less laborious and take upless physical space than field phenotyping, much larger populations canbe assayed, increasing the chances of finding a recombinant with thetarget segment from the donor line moved to the recipient line. Thecloser the linkage, the more useful the marker, as recombination is lesslikely to occur between the marker and the gene causing the trait, whichcan result in false positives. Having flanking markers decreases thechances that false positive selection will occur as a doublerecombination event would be needed. The ideal situation is to have amarker in the gene itself, so that recombination cannot occur betweenthe marker and the gene. In some embodiments, the methods disclosedherein produce a marker in a disease resistance gene, wherein the genewas identified by inferring genomic location from clustering ofconserved domains or a clustering analysis.

When a gene is introgressed by MAS, it is not only the gene that isintroduced but also the flanking regions (Gepts. (2002). Crop Sci; 42:1780-1790). This is referred to as “linkage drag.” In the case where thedonor plant is highly unrelated to the recipient plant, these flankingregions carry additional genes that may code for agronomicallyundesirable traits. This “linkage drag” may also result in reduced yieldor other negative agronomic characteristics even after multiple cyclesof backcrossing into the elite line. This is also sometimes referred toas “yield drag.” The size of the flanking region can be decreased byadditional backcrossing, although this is not always successful, asbreeders do not have control over the size of the region or therecombination breakpoints (Young et al. (1998) Genetics 120:579-585). Inclassical breeding it is usually only by chance that recombinations areselected that contribute to a reduction in the size of the donor segment(Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20backcrosses in backcrosses of this type, one may expect to find asizeable piece of the donor chromosome still linked to the gene beingselected. With markers however, it is possible to select those rareindividuals that have experienced recombination near the gene ofinterest. In 150 backcross plants, there is a 95% chance that at leastone plant will have experienced a crossover within 1 cM of the gene,based on a single meiosis map distance. Markers will allow unequivocalidentification of those individuals. With one additional backcross of300 plants, there would be a 95% chance of a crossover within 1 cMsingle meiosis map distance of the other side of the gene, generating asegment around the target gene of less than 2 cM based on a singlemeiosis map distance. This can be accomplished in two generations withmarkers, while it would have required on average 100 generations withoutmarkers (See Tanksley et al., supra). When the exact location of a geneis known, flanking markers surrounding the gene can be utilized toselect for recombinations in different population sizes. For example, insmaller population sizes, recombinations may be expected further awayfrom the gene, so more distal flanking markers would be required todetect the recombination.

The key components to the implementation of MAS are: (i) Defining thepopulation within which the marker-trait association will be determined,which can be a segregating population, or a random or structuredpopulation; (ii) monitoring the segregation or association ofpolymorphic markers relative to the trait, and determining linkage orassociation using statistical methods; (iii) defining a set of desirablemarkers based on the results of the statistical analysis, and (iv) theuse and/or extrapolation of this information to the current set ofbreeding germplasm to enable marker-based selection decisions to bemade. The markers described in this disclosure, as well as other markertypes such as SSRs and FLPs, can be used in marker assisted selectionprotocols.

SSRs can be defined as relatively short runs of tandemly repeated DNAwith lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17:6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6)Polymorphisms arise due to variation in the number of repeat units,probably caused by slippage during DNA replication (Levinson and Gutman(1987) Mol Biol Evol 4: 203-221). The variation in repeat length may bedetected by designing PCR primers to the conserved non-repetitiveflanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRsare highly suited to mapping and MAS as they are multi-allelic,codominant, reproducible and amenable to high throughput automation(Rafalski et al. (1996) Generating and using DNA markers in plants. In:Non-mammalian genomic analysis: a practical guide. Academic press. pp75-135).

Various types of SSR markers can be generated, and SSR profiles can beobtained by gel electrophoresis of the amplification products. Scoringof marker genotype is based on the size of the amplified fragment.

Various types of FLP markers can also be generated. Most commonly,amplification primers are used to generate fragment lengthpolymorphisms. Such FLP markers are in many ways similar to SSR markers,except that the region amplified by the primers is not typically ahighly repetitive region. Still, the amplified region, or amplicon, willhave sufficient variability among germplasm, often due to insertions ordeletions, such that the fragments generated by the amplificationprimers can be distinguished among polymorphic individuals, and suchindels are known to occur frequently in maize (Bhattramakki et al.(2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

SNP markers detect single base pair nucleotide substitutions. Of all themolecular marker types, SNPs are the most abundant, thus having thepotential to provide the highest genetic map resolution (Bhattramakki etal. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at aneven higher level of throughput than SSRs, in a so-called‘ultra-high-throughput’ fashion, as SNPs do not require large amounts ofDNA and automation of the assay may be straight-forward. SNPs also havethe promise of being relatively low-cost systems. These three factorstogether make SNPs highly attractive for use in MAS. Several methods areavailable for SNP genotyping, including but not limited to,hybridization, primer extension, oligonucleotide ligation, nucleasecleavage, minisequencing, and coded spheres. Such methods have beenreviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100; andBhattramakki and Rafalski (2001) Discovery and application of singlenucleotide polymorphism markers in plants. In: R. J. Henry, Ed, PlantGenotyping: The DNA Fingerprinting of Plants, CABI Publishing,Wallingford. A wide range of commercially available technologies utilizethese and other methods to interrogate SNPs including Masscode™(Qiagen), INVADER®. (Third Wave Technologies) and Invader PLUS®,SNAPSHOT®. (Applied Biosystems), TAQMAN®. (Applied Biosystems) andBEADARRAYS®. (Illumina).

A number of SNPs together within a sequence, or across linked sequences,can be used to describe a haplotype for any particular genotype (Chinget al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b),Plant Science 162:329-333). Haplotypes can be more informative thansingle SNPs and can be more descriptive of any particular genotype. Forexample, a single SNP may be allele “T” for a specific line or varietywith disease resistance, but the allele “T” might also occur in thebreeding population being utilized for recurrent parents. In this case,a haplotype, e.g. a combination of alleles at linked SNP markers, may bemore informative. Once a unique haplotype has been assigned to a donorchromosomal region, that haplotype can be used in that population or anysubset thereof to determine whether an individual has a particular gene.See, for example, WO2003054229. Using automated high throughput markerdetection platforms known to those of ordinary skill in the art makesthis process highly efficient and effective.

Many of the markers presented herein can readily be used as singlenucleotide polymorphic (SNP) markers to select for the R gene. UsingPCR, the primers are used to amplify DNA segments from individuals(preferably inbred) that represent the diversity in the population ofinterest. The PCR products are sequenced directly in one or bothdirections. The resulting sequences are aligned and polymorphisms areidentified. The polymorphisms are not limited to single nucleotidepolymorphisms (SNPs), but also include indels, CAPS, SSRs, and VNTRs(variable number of tandem repeats). Specifically, with respect to thefine map information described herein, one can readily use theinformation provided herein to obtain additional polymorphic SNPs (andother markers) within the region amplified by the primers disclosedherein. Markers within the described map region can be hybridized toBACs or other genomic libraries, or electronically aligned with genomesequences, to find new sequences in the same approximate location as thedescribed markers.

In addition to SSR's, FLPs and SNPs, as described above, other types ofmolecular markers are also widely used, including but not limited toexpressed sequence tags (ESTs), SSR markers derived from EST sequences,randomly amplified polymorphic DNA (RAPD), and other nucleic acid basedmarkers.

Isozyme profiles and linked morphological characteristics can, in somecases, also be indirectly used as markers. Even though they do notdirectly detect DNA differences, they are often influenced by specificgenetic differences. However, markers that detect DNA variation are farmore numerous and polymorphic than isozyme or morphological markers(Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

Sequence alignments or contigs may also be used to find sequencesupstream or downstream of the specific markers listed herein. These newsequences, close to the markers described herein, are then used todiscover and develop functionally equivalent markers. For example,different physical and/or genetic maps are aligned to locate equivalentmarkers not described within this disclosure but that are within similarregions. These maps may be within the species, or even across otherspecies that have been genetically or physically aligned.

In general, MAS uses polymorphic markers that have been identified ashaving a significant likelihood of co-segregation with a trait such asthe disease resistance trait. Such markers are presumed to map near agene or genes that give the plant its disease resistant phenotype, andare considered indicators for the desired trait, or markers. Plants aretested for the presence of a desired allele in the marker, and plantscontaining a desired genotype at one or more loci are expected totransfer the desired genotype, along with a desired phenotype, to theirprogeny. Thus, plants with disease resistance can be selected for bydetecting one or more marker alleles, and in addition, progeny plantsderived from those plants can also be selected. Hence, a plantcontaining a desired genotype in a given chromosomal region (i.e. agenotype associated with disease resistance) is obtained and thencrossed to another plant. The progeny of such a cross would then beevaluated genotypically using one or more markers and the progeny plantswith the same genotype in a given chromosomal region would then beselected as having disease resistance.

The SNPs could be used alone or in combination (i.e. a SNP haplotype) toselect for a favorable resistant gene allele associated with the diseaseresistance.

The skilled artisan would expect that there might be additionalpolymorphic sites at marker loci in and around a chromosome markeridentified by the methods disclosed herein, wherein one or morepolymorphic sites is in linkage disequilibrium (LD) with an allele atone or more of the polymorphic sites in the haplotype and thus could beused in a marker assisted selection program to introgress a gene alleleor genomic fragment of interest. Two particular alleles at differentpolymorphic sites are said to be in LD if the presence of the allele atone of the sites tends to predict the presence of the allele at theother site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)).The marker loci can be located within 5 cM, 2 cM, or 1 cM (on a singlemeiosis based genetic map) of the disease resistance trait QTL.

The skilled artisan would understand that allelic frequency (and hence,haplotype frequency) can differ from one germplasm pool to another.Germplasm pools vary due to maturity differences, heterotic groupings,geographical distribution, etc. As a result, SNPs and otherpolymorphisms may not be informative in some germplasm pools.

Plant Compositions

Plants identified and/or selected by any of the methods described aboveare also of interest.

Proteins and Variants and Fragments Thereof

R gene polypeptides are encompassed by the disclosure. “R genepolypeptide” and “R gene protein” as used herein interchangeably refersto a polypeptide(s) having a disease resistance activity. A variety of Rgene polypeptides are contemplated.

“Sufficiently identical” is used herein to refer to an amino acidsequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity. Insome embodiments the sequence identity is against the full lengthsequence of a polypeptide. The term “about” when used herein in contextwith percent sequence identity means+/−1.0%.

A “recombinant protein” is used herein to refer to a protein that is nolonger in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell; a protein that is expressedfrom a polynucleotide that has been edited from its native version; or aprotein that is expressed from a polynucleotide in a different genomicposition relative to the native sequence.

“Substantially free of cellular material” as used herein refers to apolypeptide including preparations of protein having less than about30%, 20%, 10% or 5% (by dry weight) of non-target protein (also referredto herein as a “contaminating protein”).

“Fragments” or “biologically active portions” include polypeptide orpolynucleotide fragments comprising sequences sufficiently identical toan R gene polypeptide or polynucleotide, respectively, and that exhibitdisease resistance when expressed in a plant.

“Variants” as used herein refers to proteins or polypeptides having anamino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identical to the parental aminoacid sequence.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a polypeptide can be preparedby mutations in the DNA. This may also be accomplished by one of severalforms of mutagenesis, such as for example site-specific double strandbreak technology, and/or in directed evolution. In some aspects, thechanges encoded in the amino acid sequence will not substantially affectthe function of the protein. Such variants will possess the desiredactivity. However, it is understood that the ability of an R genepolypeptide to confer diseaes resistance may be improved by the use ofsuch techniques upon the compositions of this disclosure.

Nucleic Acid Molecules and Variants and Fragments Thereof

Isolated or recombinant nucleic acid molecules comprising nucleic acidsequences encoding R gene polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encodingproteins with regions of sequence homology are provided. As used herein,the term “nucleic acid molecule” refers to DNA molecules (e.g.,recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell; has been editedfrom its native sequence; or is located in a different location than thenative sequence. In some embodiments, an “isolated” or “recombinant”nucleic acid is free of sequences (preferably protein encodingsequences) that naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For purposes of thedisclosure, “isolated” or “recombinant” when used to refer to nucleicacid molecules excludes isolated chromosomes. For example, in variousembodiments, the recombinant nucleic acid molecules encoding R genepolypeptides can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived.

In some embodiments an isolated nucleic acid molecule encoding R genepolypeptides has one or more change in the nucleic acid sequencecompared to the native or genomic nucleic acid sequence. In someembodiments the change in the native or genomic nucleic acid sequenceincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; changes in the nucleic acidsequence due to the amino acid substitution, insertion, deletion and/oraddition compared to the native or genomic sequence; removal of one ormore intron; deletion of one or more upstream or downstream regulatoryregions; and deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence. In some embodiments the nucleicacid molecule encoding an R gene polypeptide is a non-genomic sequence.

A variety of polynucleotides that encode R gene polypeptides or relatedproteins are contemplated. Such polynucleotides are useful forproduction of R gene polypeptides in host cells when operably linked toa suitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides that encode Rgene polypeptides or related proteins.

“Complement” is used herein to refer to a nucleic acid sequence that issufficiently complementary to a given nucleic acid sequence such that itcan hybridize to the given nucleic acid sequence to thereby form astable duplex. “Polynucleotide sequence variants” is used herein torefer to a nucleic acid sequence that except for the degeneracy of thegenetic code encodes the same polypeptide.

In some embodiments the nucleic acid molecule encoding the R genepolypeptide is a non-genomic nucleic acid sequence. As used herein a“non-genomic nucleic acid sequence” or “non-genomic nucleic acidmolecule” or “non-genomic polynucleotide” refers to a nucleic acidmolecule that has one or more change in the nucleic acid sequencecompared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; optimization of the nucleic acidsequence for expression in plants; changes in the nucleic acid sequenceto introduce at least one amino acid substitution, insertion, deletionand/or addition compared to the native or genomic sequence; removal ofone or more intron associated with the genomic nucleic acid sequence;insertion of one or more heterologous introns; deletion of one or moreupstream or downstream regulatory regions associated with the genomicnucleic acid sequence; insertion of one or more heterologous upstream ordownstream regulatory regions; deletion of the 5′ and/or 3′ untranslatedregion associated with the genomic nucleic acid sequence; insertion of aheterologous 5′ and/or 3′ untranslated region; and modification of apolyadenylation site. In some embodiments the non-genomic nucleic acidmolecule is a synthetic nucleic acid sequence.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding R gene polypeptides are also encompassed by theembodiments. “Fragment” as used herein refers to a portion of thenucleic acid sequence encoding an R gene polypeptide. A fragment of anucleic acid sequence may encode a biologically active portion of an Rgene polypeptide or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Nucleicacid molecules that are fragments of a nucleic acid sequence encoding anR gene polypeptide comprise at least about 150, 180, 210, 240, 270, 300,330, 360, 400, 450, or 500 contiguous nucleotides or up to the number ofnucleotides present in a full-length nucleic acid sequence encoding a Rgene polypeptide identified by the methods disclosed herein, dependingupon the intended use. “Contiguous nucleotides” is used herein to referto nucleotide residues that are immediately adjacent to one another.Fragments of the nucleic acid sequences of the embodiments will encodeprotein fragments that retain the biological activity of the R genepolypeptide and, hence, retain disease resesistance. “Retains diseaseresistance” is used herein to refer to a polypeptide having at leastabout 10%, at least about 30%, at least about 50%, at least about 70%,80%, 90%, 95% or higher of the disease resistance of the full-length Rgene polypeptide.

“Percent (%) sequence identity” with respect to a reference sequence(subject) is determined as the percentage of amino acid residues ornucleotides in a candidate sequence (query) that are identical with therespective amino acid residues or nucleotides in the reference sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyamino acid conservative substitutions as part of the sequence identity.Alignment for purposes of determining percent sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences (e.g., percentidentity of query sequence=number of identical positions between queryand subject sequences/total number of positions of query sequence×100).

The embodiments also encompass nucleic acid molecules encoding R genepolypeptide variants. “Variants” of the R gene polypeptide encodingnucleic acid sequences include those sequences that encode the R genepolypeptides identified by the methods disclosed herein, but that differconservatively because of the degeneracy of the genetic code as well asthose that are sufficiently identical as discussed above. Naturallyoccurring allelic variants can be identified with the use of well-knownmolecular biology techniques, such as polymerase chain reaction (PCR)and hybridization techniques as outlined below. Variant nucleic acidsequences also include synthetically derived nucleic acid sequences thathave been generated, for example, by using site-directed mutagenesis butwhich still encode the R gene polypeptides disclosed herein.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded R gene polypeptides,without altering the biological activity of the proteins. Thus, variantnucleic acid molecules can be created by introducing one or morenucleotide substitutions, additions and/or deletions into thecorresponding nucleic acid sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer activity to identify mutants that retain activity.Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional polypeptide homologues and fragments thereof withdesired properties. A variety of such reactions are known. Methods forproducing a variant of any nucleic acid listed herein comprisingrecursively recombining such polynucleotide with a second (or more)polynucleotide, thus forming a library of variant polynucleotides arealso embodiments of the disclosure, as are the libraries produced, thecells comprising the libraries and any recombinant polynucleotideproduced by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onactivity, as is wherein such recursive recombination is done in vitro orin vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.such activity at a desired pH, etc. This can include identifying anyactivity that can be detected, for example, in an automated orautomatable format, by any of the assays in the art. A variety ofrelated (or even unrelated) properties can be evaluated, in serial or inparallel, at the discretion of the practitioner.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from a different source. In this manner, methodssuch as PCR, hybridization, and the like can be used to identify suchsequences based on their sequence homology to the sequences identifiedby the methods disclosed herein. Sequences that are selected based ontheir sequence identity to the entire sequences set forth herein or tofragments thereof are encompassed by the embodiments. Such sequencesinclude sequences that are orthologs of the sequences. The term“orthologs” refers to genes derived from a common ancestral gene andwhich are found in different species as a result of speciation. Genesfound in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences sharesubstantial identity as defined elsewhere herein.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

In hybridization methods, all or part of the nucleic acid sequence canbe used to screen cDNA or genomic libraries. Methods for construction ofsuch cDNA and genomic libraries are generally known in the art and aredisclosed in Sambrook and Russell, (2001), supra. The so-calledhybridization probes may be genomic DNA fragments, cDNA fragments, RNAfragments or other oligonucleotides and may be labeled with a detectablegroup such as 32P or any other detectable marker, such as otherradioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor.Probes for hybridization can be made by labeling syntheticoligonucleotides based on the known polypeptide-encoding nucleic acidsequences disclosed herein. Degenerate primers designed on the basis ofconserved nucleotides or amino acid residues in the nucleic acidsequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequences encoding polypeptides or afragment or variant thereof. Methods for the preparation of probes forhybridization and stringency conditions are generally known in the artand are disclosed in Sambrook and Russell, (2001), supra.

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructs,particularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides, may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant cells, bacteria, yeast, baculovirus,protozoa, nematodes and algae. The transformed organism comprises a DNAmolecule of the embodiments, an expression cassette comprising the DNAmolecule or a vector comprising the expression cassette, which may bestably incorporated into the genome of the transformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the polypeptide gene sequence of the disclosure to beunder the transcriptional regulation of the regulatory regions. The DNAconstruct may additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments the DNA construct comprises a polynucleotideencoding an R gene polypeptide of the embodiments. In some embodimentsthe DNA construct comprises a polynucleotide encoding a fusion proteincomprising an R gene polypeptide of the embodiments.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464)), the omega enhancer or theomega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNAed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may alsobe used. The above list of transcriptional enhancers is not meant to belimiting. Any appropriate transcriptional enhancer can be used in theembodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred usage. For example, although nucleic acid sequences ofthe embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific preferences and GC content preferences of monocotyledons ordicotyledons as these preferences have been shown to differ (Murray etal. (1989) Nucleic Acids Res. 17:477-498). Thus, the plant-preferred fora particular amino acid may be derived from known gene sequences fromplants.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism.

In some aspects, a DNA construct may encode a double stranded RNAtargeting an effector protein transcript, producing a reduction ineffector protein translation. In some embodiments, the reduction ineffector protein produces a disease resistance phenotype in a plant orplant cell. A wide variety of eukaryotic organisms, including plants,animals, and fungi, have evolved several RNA-silencing pathways toprotect their cells and genomes against invading nucleic acids, such asviruses or transposons, and to regulate gene expression duringdevelopment or in response to external stimuli (for review, seeBaulcombe (2005) Trends Biochem. Sci. 30:290-93; Meins et al. (2005)Annu. Rev. Cell Dev. Biol. 21:297-318). In plants, RNA-silencingpathways have been shown to control a variety of developmental processesincluding flowering time, leaf morphology, organ polarity, floralmorphology, and root development (reviewed by Mallory and Vaucheret(2006) Nat. Genet. 38:S31-36). All RNA-silencing systems involve theprocessing of double-stranded RNA (dsRNA) into small RNAs of 21 to 25nucleotides (nt) by an RNaseIII-like enzyme known as Dicer or Dicer-likein plants (Bernstein et al. (2001) Nature 409:363-66; Xie et al. (2004)PLOS Biol. 2 E104:0642-52; Xie et al. (2005) Proc. Natl. Acad. Sci. USA102:12984-89; Dunoyer et al. (2005) Nat. Genet. 37:1356-60). These smallRNAs are incorporated into silencing effector complexes containing anArgonaute protein (for review, see Meister and Tuschl (2004) Nature431:343-49).

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide(s) or polypeptide(s) gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide(s) or polypeptide(s) into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Lec1 transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988)Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens).

Methods to Introduce Genome Editing Technologies into Plants

In some embodiments, polynucleotide compositions can be introduced intothe genome of a plant using genome editing technologies, or previouslyintroduced polynucleotides in the genome of a plant may be edited usinggenome editing technologies. For example, the identified polynucleotidescan be introduced into a desired location in the genome of a plantthrough the use of double-stranded break technologies such as TALENs,meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Forexample, the identified polynucleotides can be introduced into a desiredlocation in a genome using a CRISPR-Cas system, for the purpose ofsite-specific insertion. The desired location in a plant genome can beany desired target site for insertion, such as a genomic region amenablefor breeding or may be a target site located in a genomic window with anexisting trait of interest. Existing traits of interest could be eitheran endogenous trait or a previously introduced trait.

In some embodiments, where an R allele has been identified in a genome,genome editing technologies may be used to alter or modify thepolynucleotide sequence. Site specific modifications that can beintroduced into the desired R gene allele polynucleotide include thoseproduced using any method for introducing site specific modification,including, but not limited to, through the use of gene repairoligonucleotides (e.g. US Publication 2013/0019349), or through the useof double-stranded break technologies such as TALENs, meganucleases,zinc finger nucleases, CRISPR-Cas, and the like. Such technologies canbe used to modify the previously introduced polynucleotide through theinsertion, deletion or substitution of nucleotides within the introducedpolynucleotide. Alternatively, double-stranded break technologies can beused to add additional nucleotide sequences to the introducedpolynucleotide. Additional sequences that may be added include,additional expression elements, such as enhancer and promoter sequences.In another embodiment, genome editing technologies may be used toposition additional disease resistant proteins in close proximity to theR gene polynucleotide compositions within the genome of a plant, inorder to generate molecular stacks disease resistant proteins.

An “altered target site,” “altered target sequence.” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

In some embodiments, an effector sequence is used to survey a plant pestpopulation to identify allele diversity and allele frequency of theeffector in a field pest population. In further embodiments, a plantcomprising an R-gene that interacts with the effector is deployed basedon the allele specific data. In some embodiments, the effector sequencecomprises any one of SEQ ID NO: 1626-1628, or 1631.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed subject matter. It is understood that the examples andembodiments described herein are for illustrative purposes only, andpersons skilled in the art will recognize various reagents or parametersthat can be altered without departing from the spirit of the disclosureor the scope of the appended claims.

Example 1. Identify Putative Pathogen Effectors

A list of putative Puccinia polysora effectors were generated bycomputational analysis. RNA-seq reads from Puccinia polysora infectedmaize leaves were mapped against the Puccinia polysora genome withTophat2 using the default parameters (See Kim, D., Pertea, G., Trapnell,C. et al. TopHat2: accurate alignment of transcriptomes in the presenceof insertions, deletions and gene fusions. Genome Biol 14, R36 (2013)doi:10.1186/gb-2013-14-4-r36). Alignments were then passed to Stringtieto assemble transcript models using default parameters (See Pertea, M.,Pertea, G., Antonescu, C. et al. StringTie enables improvedreconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33,290-295 (2015) doi:10.1038/nbt.3122). Transcript models were furthersupplemented with a PacBio Iso-seq library from germinating Pucciniapolysora spores. Using custom Python scripts, the longest possibleprotein was predicted from all transcript models. The resulting proteinswere analyzed for features associated with pathogen effectors utilizingthe following criteria and software: presence of signal peptide(SignalP, see Nielsen H. (2017) Predicting Secretory Proteins withSignalP. In: Kihara D. (eds) Protein Function Prediction. Methods inMolecular Biology, vol 1611. Humana Press, New York, N.Y.), lack oftransmembrane domain (TMHMM, see Anders Krogh, Bjorn Larsson, Gunnar vonHeijne, Erik L. L Sonnhammer, Predicting transmembrane protein topologywith a hidden markov model: application to complete genomes11Edited byF. Cohen, Journal of Molecular Biology, Volume 305, Issue 3, 2001, Pages567-580), small size (custom Python script), small effector-associatedmotifs (custom Python script), conserved effector-associated domains(HMMer, see Reddy, S. Multiple Alignment Using Hidden Markov Models.ISMB-95 Proceedings, pp. 114-120 (1995)), expression in infected maizeleaf samples (Salmon, see Patro R, Duggal G, Love M I, Irizarry R A,Kingsford C. Salmon provides fast and bias-aware quantification oftranscript expression. Nat Methods. 2017; 14(4):417-419.doi:10.1038/nmeth.4197), as well as a machine learning-based prediction(EffectorP, see Sperschneider, J., Gardiner, D. M., Dodds, P. N., Tini,F., Covarelli, L., Singh, K. B., Manners, J. M. and Taylor, J. M.(2016), EffectorP: predicting fungal effector proteins from secretomesusing machine learning. New Phytol, 210: 743-761.doi:10.1111/nph.13794). The putative effectors identified in FIG. 3 areused for the subsequent screening.

Example 2. Generate Effector Over-Expression Vectors

The signal peptide sequence was predicted for each putative Pucciniapolysora effector and trimmed off the sequence. The effector sequencesare optimized for expression in maize, based on codon usage preferenceand GC content. The maize codon-optimized sequences are then synthesizedand cloned into vectors containing two expression cassettes, onecontaining the effector sequence and another containing the luciferasereporter gene. Both the effector and luciferase are under the control ofstrong constitutive promoters.

Example 3. Identify and Confirm Effectors Recognized Disease ResistanceGenes

A protoplast-based system is used to screen for effectors which cancause rapid protoplast death (indicated by a significant reduction inluciferase activity, presumably caused by HR).

Maize seeds are sown and watered in flats of a propagation mix andallowed to germinate under light for four to six days at 26° C. Aftershoots emerged, flats are transferred to dark for four to six days.Plants are ready for protoplast isolation after the second leaf is about10-15 cm above the first leaf.

For each plant, the green tip of the second leaf (about 1-2 cm) isremoved. The next 6-8 cm of the leaf blade is collected. A stack 10-15leaves is cut (with a new, clean razor blade) into 0.5 mm stripeswithout bruising or wounding the leaves. The leaf strips are immediatelysubmerged into a digestion solution. The leaves are vacuum infiltratedin the digestion solution for 30 minutes at room temperature (RT). Thedigestion is continued without vacuum for two hours with gentle shaking(40 RPM) on a platform shaker. The shaking is increased to 60 RPM for 10minutes to release the protoplasts into the solution.

The digestion solution containing protoplasts is filtered through anylon mesh (40 μm), and washed with mannitol solution. The solution isthen centriguged at 70×g for 3 minutes to pellet the protoplasts, andremove as much of the supernatant as possible. The protoplasts arewashed with mannitol solution and centrifuged two more times. After thelast wash, the protoplasts are resuspended in a small amount of MMgsolution (5-10 mL). Sample yield is calculated using a hemocytometer oran automated cell counter.

20 uL of plasmid DNA is added to 200 uL protoplast solution and mixedgently, then added 220 ul of PEG solution. The mixture is incubated atroom temperature for about 15 minutes. The PEG reaction is diluted with1 mL of WI solution, and mixed and stop the transfection. The mixture iscentrifuged at 100×g for two minutes and the supernatant is removed. Theprotoplasts are resuspended and incubated overnight.

After 16 and 40 hours of incubation, protoplasts are resuspended insolution by inverting the microfuge tube or by gentle pipetting. 50 μlof Promega Steady-Glo are added to 50 μl of protoplast solution andallowed to mix for at least 5 minutes. Luciferase luminescence isquantified using the Steady-Glo program on a Promega GloMax Explorermicroplate reader.

Each effector construct is introduced into protoplasts from Inbred A.One of the putative effectors tested, Effector 32 (SEQ ID NO: 2), wasidentified and confirmed to induce cell death in Inbred A, whichcontains the SCR resistance gene NLR03 (SEQ ID NO: 9). Effector 32 alsoinduced cell death in CML496. When protoplasts from a susceptible linewere co-transfected with both NLR03 and Effector 32, a similar celldeath was observed. NLR03 recognized Effector 32 in both Inbred A andCML496, which triggers a hypersensitive response (“HR”) and results in acell death resistance response (See Tables 1 and 2).

TABLE 1 Effector 32 triggers HR in Inbred A protoplasts Maize GenotypeGenes Average luciferase reads Inbred A AvrSr35 1,385,777.78 Inbred AAvrSr35 + Sr35 10,763.89 Inbred A GFP 2,506,000.00 Inbred A SCREffector32 64,527.78

TABLE 2 Effector 32 triggers HR in CML496 protoplasts Maize GenotypeGenes Average luciferase reads CML496 AvrSr35 1,397,666.67 CML496AvrSr35 + Sr35 10,315.78 CML496 GFP 2,171,111.11 CML496 SCR Effector3296,004.44

Example 4: Confirm Recognition and Interaction of Between PucciniaPolvsora Effector 32 and NLR03

Recognition of pathogen effectors by R proteins leads toeffector-triggered immunity (ETI), often culminating in a HR cell deathaccompanied with ROS production (Jones and Dangl, 2006).Agrobacterium-mediated transient expression in tobacco plant (Li, 2011)was used to confirm the recognition of Effector 32 by NLR03. Effector 32and NLR03 were cloned into a binary vector and introduced intoAgrobacterium tumefaciens. Sr35 and AvrSr35 were used as the positivecontrol. HR cell death (visual) and accumulated H₂O₂ (DAB staining) wereobserved when Effector 32 and NLR03 were co-expressed in N. benthamiana.Further co-immunoprecipitation (Co-IP) and yeast two hybrid (Y2H) wereperformed to check the physical interaction between Effector 32 andNLR03. The binding of Effector 32 and NLR03 was detected in Co-IP assaybut not in Y2H, indicating Effector 32 directly interacts with NLR03 inplanta, and the interaction may require other proteins.

TABLE 3 Co-transfection of NLR3 and Effector 32 triggers HR in PHR03protoplasts Maize Genotype Genes Average luciferase reads PHR03 AvrSr353,277,555.56 PHR03 AvrSr35 + Sr35 97,661.11 PHR03 GFP + NLR33,715,111.11 HC69 SCR Effector 32 6,104,444.44 PHR03 SCR Effector32 +NLR3 154,711.11

Example 5: Allele-Specific Recognition of Effector 32 by NLR03

Ninety-two samples from Puccinia polysora-infected maize leaves werecollected from multiple locations in China and the US. Effector 32 wasPCR amplified and re-sequenced from the samples. After trimming away thesignal peptide, a total of 15 distinct Effector 32 alleles wereidentified. Each of the Effector 32 allele was transfected alone orco-transfected with NLR03 into the Susceptible line protoplasts. Eightof the Effector 32 alleles reduce the luciferase activity by greaterthan 90%, while three of the alleles cause a less than 60% reduction(Table 4). The results suggest that while NLR03 can trigger differentlevels of HR by different Effector 32 alleles, and NLR03 may conferdifferent level of resistance to isolates containing different Effector32 alleles. The different intensity of immune responses caused bydifferent Effector 32 alleles has been confirmed in N. benthamiana withselected Effector 32 alleles. Surveying effector alleles and allelefrequency in a particular pathogen population would predict if aspecific resistance gene is efficacious in providing resistance.

TABLE 4 Effector 32 allele activity Protein sequence 40 hour Effector(without Signal CML496 alleles Peptide) reduction % Effector32-OR SEQ IDNO: 4 95 Effector32-A SEQ ID NO: 10 92 Effector32-C SEQ ID NO: 11 93Effector32-E SEQ ID NO: 12 98 Effector32-F SEQ ID NO: 13 91 Effector32-GSEQ ID NO: 14 22 Effector32-J SEQ ID NO: 15 93 Effector32-M SEQ ID NO:16 82 Effector32-N SEQ ID NO: 17 55 Effector32-O SEQ ID NO: 18 58Effector32-Q SEQ ID NO: 19 85 Effector32-R SEQ ID NO: 20 86 Effector32-VSEQ ID NO: 21 97 Effector32-W SEQ ID NO: 22 98 Effector32-X SEQ ID NO:23 72

Example 6: Use Effector 32 to Characterize SCR Resistance Donors

Effector 32 was transfected into protoplasts from 30 SCR resistancemaize inbred lines. Effector 32 triggered HR in 6 inbred lines, inaddition to Inbred A and CML496, suggesting that these 6 lines containthe same or similar resistance genes which can recognize Effector 32,and the mechanism of resistance is similar among these lines. Effectorsmay be used to characterize diverse resistance donors and understandtheir mechanisms of resistance.

RNA-seq data from CML415 indicate that CML415 has a NLR (SEQ ID NO: 9,cDNA SEQ ID NO: 8) with 99.2% identity to NLR03 (SEQ ID NO: 7), cDNA SEQID NO: 6). Co-expressing the CML415 NLR gene and Effector 32 in maizeprotoplasts caused protoplast death, indicating the CML415 NLR gene canrecognize Effector 32 and confer resistance to SCR.

Example 7. Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with an identified Rgene sequence of the disclosure, the method of Zhao is employed (U.S.Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contentsof which are hereby incorporated by reference). Briefly, immatureembryos are isolated from maize and the embryos contacted with asuspension of Agrobacterium under conditions whereby the bacteria arecapable of transferring the regulatory element sequence of thedisclosure to at least one cell of at least one of the immature embryos(step 1: the infection step). In this step the immature embryos areimmersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). The immature embryosare cultured on solid medium following the infection step. Following theco-cultivation period an optional “resting” step is performed. In thisresting step, the embryos are incubated in the presence of at least oneantibiotic known to inhibit the growth of Agrobacterium without theaddition of a selective agent for plant transformants (step 3: restingstep). Next, inoculated embryos are cultured on medium containing aselective agent and growing transformed calli are recovered (step 4: theselection step). Plantlets are regenerated from the calli (step 5: theregeneration step) prior to transfer to the greenhouse.

Example 8. Editing R Genes Target Site Selection

The gRNA/Cas9 Site directed nuclease system, described in WO2015026885,WO20158026887, WO2015026883, and WO2015026886 (each incorporated hereinby reference), is to edit the R gene by replacing a native allele with aresistant allele. Pairs of target sites are used for removing the entireR allele from the target line, including the predicated promoter, thecoding sequence, and 1 kb of 3′ UTR. The DNA repair template isco-delivered with Cas9 and guide RNA plasmids.

Cas9 Vector Construction

A Cas9 gene from Streptococcus pyogenes M1 GAS (SF370) is maize codonoptimized per standard techniques known in the art, and the potatoST-LS1 intron is introduced in order to eliminate its expression in E.coli and Agrobacterium. To facilitate nuclear localization of the Cas9protein in maize cells, the Simian virus 40 (SV40) monopartite aminoterminal nuclear localization signal is incorporated at the aminoterminus of the Cas9 open reading frame. The maize optimized Cas9 geneis operably linked to a maize Ubiquitin promoter using standardmolecular biological techniques. In addition to the amino terminalnuclear localization signal SV40, a C-terminal bipartitite nuclearlocalization signal from Agrobacterium tumefaciens VirD2 endonucleasewas fused at the end of exon 2. The resulting sequence includes the Zeamays ubiquitin promoter, the 5′ UTR of the ZM-ubiquitin gene, intron 1of the ZM-ubiquitin gene, the SV40 nuclear localization signal, Cas9exon 1 (ST1), the potato-LS1 intron, Cas9 exon 2 (ST1), the VirD2endonuclease nuclear localization signal, and the pinII terminator.

Guide RNA Vector Construction

To direct Cas9 nuclease to the designated genomic target sites, a maizeU6 polymerase III promoter (see WO2015026885, WO20158026887,WO2015026883, and WO2015026886) and its cognate U6 polymerase IIItermination sequences (TTTTTTTT) are used to direct initiation andtermination of gRNA expression. Guide RNA variable targeting domains forR gene editing are identified, which correspond to the genomic targetsites. Oligos containing the DNA encoding each of the variablenucleotide targeting domains are synthesized and cloned into a gRNAexpression cassette. Each guide RNA expression cassette consists of theU6 polymerase III maize promoter operably linked to one of the DNAversions of the guide RNA followed by the cognate U6 polymerase IIItermination sequence. The DNA version of the guide RNA consists of therespective nucleotide variable targeting domain followed by apolynucleotide sequence capable of interacting with the double strandbreak inducing endonuclease.

Repair Template Vector Construction

The substitution/replacement template for CR6/CR9 contains the resistantallele of the R gene, and the substitution template contains the sameresistant allele of the R gene and the homology sequences flanking the5′ and 3′ in the target line. The homology arm sequences are synthesizedand then cloned with substitutive R gene genomic sequences via astandard seamlessness Gibson cloning method.

Delivery of the Guide RNA/Cas9 Endonuclease System DNA to Maize

Plasmids containing the Cas9 and guide RNA expression cassettesdescribed above are co-bombarded with plasmids containing thetransformation selectable marker NPTII and the transformation enhancingdevelopmental genes ODP2 (AP2 domain transcription factor ODP2 (Ovuledevelopment protein 2)) and Wuschel (20151030-6752 USPSP) into elitemaize lines' genomes. Transformation of maize immature embryos may beperformed using any method known in the art or the method describedbelow.

In one transformation method, ears are husked and surface sterilized in30-50% Clorox bleach plus 0.5% Micro detergent for 10 minutes and thenrinsed two times with sterile water. The immature embryos are isolatedand placed embryo axis side down (scutellum side up), with 25 embryosper plate, on 13224E medium for 2-4 hours and then aligned within the2.5-cm target zone in preparation for bombardment.

DNA of plasmids is adhered to 0.6 μm (average diameter) gold pelletsusing a proprietary lipid-polymer mixture of TransIT®-2020 (Cat #MIR5404, Minis Bio LLC, Madison, Wis. 5371). A DNA solution was preparedusing 1 μg of plasmid DNA and optionally, other constructs were preparedfor co-bombardment using 10 ng (0.5 μl) of each plasmid. To thepre-mixed DNA, 50 μl of prepared gold particles (30 mg/ml) and 1 μlTransIT®-2020 are added and mixed carefully. The final mixture isallowed to incubate under constant vortexing at low speed for 10minutes. After the precipitation period, the tubes are centrifugedbriefly, and liquid is removed. Gold particles are pelleted in amicrofuge at 10,000 rpm for 1 min, and aqueous supernatant is removed.120 μl of 100% EtOH is added, and the particles are resuspended by briefsonication. Then, 10 μl is spotted on to the center of each macrocarrierand allowed to dry about 2 minutes before bombardment, with a totaloften aliquots taken from each tube of prepared particles/DNA.

The sample plates are bombarded with a Biolistic PDA-1000/He (Bio-Rad).Embryos are 6 cm from the macrocarrier, with a gap of ⅛th of an inchbetween the 200 psi rupture disc and the macrocarrier. All samplesreceive a single shot.

Following bombardment, the embryos are incubated on the bombardmentplate for ˜20 hours then transferred to 13266L (rest/induction medium)for 7-9 days at temperatures ranging from 26-30° C. Embryos are thentransferred to the maturation media 289H for ˜21 days. Mature somaticembryos are then transferred to germination media 272G and moved to thelight. In about 1 to 2 weeks plantlets containing viable shoots androots are sampled for analysis and sent to the greenhouse where they aretransferred to flats (equivalent to a 2.5″ pot) containing potting soil.After 1-2 weeks, the plants are transferred to Classic 600 pots (1.6gallon) and grown to maturity.

Media:

Bombardment medium (13224E) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 190.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H2O following adjustment to pH 5.8 withKOH); 6.3 g/l Sigma agar (added after bringing to volume with D-I H2O);and 8.5 mg/l silver nitrate (added after sterilizing the medium andcooling to room temperature).

Selection medium (13266L) comprises 1650 mg/l ammonium Nitrate, 277.8mg/l ammonium Sulfate, 5278 mg/l potassium nitrate, calcium chloride,anhydrous 407.4 mg/l calcium chloride, anhydrous, 234.92 mg/l magnesiumsulfate, anhydrous, 410 mg/l potassium phosphate, monobasic, 8 mg/lboric acid, 8.6 mg/l, zinc sulfate.7h2o, 1.28 mg/l potassium iodide,44.54 mg/l ferrous sulfate.7h2o, 59.46 mg/l na2edta.2h2o, 0.025 mg/lcobalt chloride.6h2o, 0.4 mg/l molybdic acid (sodium salt).2h2o, 0.025mg/l cupric sulfate.5h2o, 6 mg/l manganese sulfate monohydrate, 2 mg/lthiamine, 0.6 ml/l b5h minor salts 1000×, 0.4 ml/l eriksson's vitamins1000×, 6 ml/l s&h vitamin stock 100×, 1.98 g/l 1-proline, 3.4 mg/lsilver nitrate, 0.3 g/l casein hydrolysate (acid), 20 g/l sucrose, 0.6g/l glucose, 0.8 mg/l 2.4-d, 1.2 mg/l dicamba, 6 g/l tc agar, 100 mg/lagribio carbenicillin, 25 mg/l cefotaxime, and 150 mg/l geneticin (g418)

Plant regeneration medium (289H) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 8.0 g/l Sigma agar (addedafter bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acidand 150 mg/l Geneticin (G418) (added after sterilizing the medium andcooling to 60° C.).

Hormone-free medium (272G) comprises 4.3 g/l MS salts (GIBCO 11117-074),5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/lthiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought tovolume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/lsucrose (brought to volume with polished D-I H2O after adjusting pH to5.6); and 0.5 mg/l IBA and 150 mg/l Geneticin (G418) and 6 g/lbacto-agar (added after bringing to volume with polished D-I H2O),sterilized and cooled to 60° C.

Screening of T0 Plants and Event Characterization

To identify swap positive events, PCR is performed using SigmaExtract-N-Amp PCR ready mix. PCR is performed to assay the 5′ junctionusing a primer pair of the R gene, while primary PCR with a primer pairwas combined with secondary allele differentiation qPCR to screen the 3′junction due to high homology of the intended edited variants and theunmodified genomic sequence.

T1 Analysis

The allele swap variants are transferred to a controlled environment.Pollen from T0 plants is carried to recurrent parent plants to produceseed. T1 plants are put through more comprehensive molecularcharacterization to not only confirm that swaps observed in TO plant arestably inherited but also to verify that the T1 or later generationplants are free from any foreign DNA elements used during thetransformation process. First, qPCR is performed on all helper genesincluding Cas9, the guide RNAs, the transformation selection marker(NPTII), and the transformation enhancing genes ODP2 and WUS2 to makesure the genes segregated away from the generated mutant alleles. The T1plants are sampled using Southern by Sequencing (SbS) analysis tofurther demonstrate that the plants are free of any foreign DNA.

Example 9. Screening of Plants for Disease Resistance Gray Leaf Spot

The plants are inoculated with Cercospora zeae-maydis in the greenhouseand/or field. Disease scoring is done by rating plants on a 1-9 scalewith 1 as the worst and 9 as the best. Check inbreds and/or hybrids withknown disease response are used as a guide for the best time to scoreand for rating calibration. Flowering data is also taken by noting thedate on which 50% of each plant showed silks and converting this to agrowing degree heat unit score (GDUSLK) based upon weather data at thatlocation.

Anthracnose Stalk Rot

The plants are grown and evaluated for response to Cg (Colletotrichumgraminicola). Plants evaluated for resistance to Cg in the greenhouseand/or by inoculating with Cg. Late in the growing stage, the stalks aresplit and the progression of the disease is scored by observation of thecharacteristic black color of the fungus as it grows up the stalk.Disease ratings are conducted as described by Jung et al. (1994)Theoretical and Applied Genetics, 89:413-418). The total number ofinternodes discolored greater than 75% (antgr75) are recorded on thefirst five internodes (See FIG. 20 ). This provided a disease scoreranging from 0 to 5, with zero indicating no internodes more than 75%discolored and 5 indicating complete discoloration of the first fiveinternodes. The center two plots are harvested via combine atphysiological maturity and grain yield in kg/ha was determined.

Northern Leaf Blight

Plants are tested in greenhouse and/or field experiments for efficacyagainst the northern leaf blight pathogen (Exserohilum turcicum). Plantsare challenged with the pathogen for which the R genes are thought toprovide resistance. Plants are scored as resistant or susceptible basedon disease symptoms; in the field, plants will be scored on a 1-9 scalewhere 9 is very resistant and 1 is very susceptible.

Head Smut

The sori containing teliospores of S. reliana are collected from thefield in the previous growing season and stored in cloth bag in a dryand well ventilated environment. Before planting, spores are removedfrom the sori, filtered, and then mixed with soil at a ratio of 1:1000.The mixture of soil and teliospores are used to cover maize kernels whensowing seeds to conduct artificial inoculation. Plants at maturity stageare scored for the presence/absence of sorus in either ear or tassels asan indicator for susceptibility/resistance.

Example 10. Identification and Confirmation of Putative Exserohilumturcicum Effectors Recognized by PH26N_NLB18

For E. turcicum effector discovery, RNA-seq from leaves infected with E.turcicum were mapped against its genome. The same criteria as describedabove in Example 1 was employed to discover effectors based on the genemodels obtained. Computational genomics was then employed to identifyputative effectors that were present in E. turcicum race 0 and race 1,which can be controlled by PH26N_NLB18 (a previously identified NLBresistance gene, see US Application Publication Number 2015-0376644 A1,herein incorporated by reference), but were either absent or containednon-synonymous mutations in race23N, which cannot be controlled byPH26N_NLB18. Four effectors were selected from the comparative analysisfor further screening and validation.

The Agrobacterium-mediated transient expression system in Nicotianabenthamiana was used to screen for effectors which can trigger rapidcell death (HR) when co-expressed with PH26N_NLB18. The coding sequencesPH26N_NLB18 and four putative effectors (NLB_031, 032, 033, 034) weresynthesized and cloned into a binary vector, under the control of thesoy Ubi promoter. The resulting constructs were introduced into A.tumefaciens strain AGL1 by electroporation (Hellens et al., 2000) andtransformants were selected with kanamycin (50 mg/mL). For theinfiltration, recombinant strains of A. tumefaciens were prepared andinfiltrated into tobacco leaves as describe by Yang et al. (2000).Symptoms were scored daily, and pictures were taken 2-3 days afterinfiltration.

HR cell death (visual) and accumulated H₂O₂ (DAB staining) were onlyobserved when PH26N_NLB18 and NLB_034 (SEQ ID NO: 1623) wereco-expressed in N. benthamiana. Further co-immunoprecipitation (Co-IP)and yeast two hybrid (Y2H) were performed to examine the physicalinteraction between PH26N_NLB18 and NLB_034. The interaction betweenPH26N_NLB18 and NLB_034 was confirmed by both Co-IP assay and Y2H,indicating PH26N_NLB18 was able to recognize and directly interact withNLB_034 in planta to initiate immune responses.

Example 12: Allele-Specific Recognition of E. turcicum Effector NLB_034by PH26N_NLB18

After trimming away the signal peptide, 3 distinct NLB_034 alleles,NLB_034A (the original allele), NLB_034B and NLB_034C, were identifiedfrom E. turcicum Races 0, 1 and 23N. Each of the NLB_034 alleles wasco-expressed with PH26N_NLB18 in N. benthamiana leaves for cell deathobservation. While both NLB_034A and NLB_034B alleles could berecognized by PH26N_NLB18, the NLB_034C allele couldn't trigger HR celldeath when co-expressed with PH26N_NLB18. Also, NLB_034C didn't interactwith PH26N_NLB18 in both Co-IP and Y2H assays. The allele-specificrecognition implies race-specific resistance by PH26N_NLB18. Table 5shows the sequences identified as effectors.

TABLE 5 NLB Effector Sequences SEQ ID NO: Sequence Description 1620NLB_034A CDS 1621 NLB_034B CDS 1622 NLB_034C CDS 1623 NLB_034A Genomic1624 NLB_034B Genomic 1625 NLB_034C Genomic 1626 NLB_034A Protein 1627NLB_034B Protein 1628 NLB_034C Protein

Example 13. Identification and Confirmation of Putative Effectors inColletotrichum graminicola

The same effector discovery methods were employed to discover C.graminicola effectors, utilizing RNA-seq reads from maize leavesinfected with C. graminicola mapped against its genome to obtain genemodels. After employing the same computational criteria as describedabove (see Example 1), the top 178 effectors utilized for subsequentscreening.

A pair of NLRs from maize, Rcg and Rcg1b, were previously identified tomediate resistance to the fungal pathogen C. graminicola (US ApplicationPublication Number 2018-0112280 A1). Since Rcg1b contain an integrateddomain (ID), it was hypothesized that the ID recognizes effectorprotein(s) via direct interaction. The Rcg1b ID was used as a bait toscreen the 178 predicted effectors via Y2H. The coding sequence of theRcg1b ID and the 178 putative effectors (ANTROT_1-178) were synthesizedand cloned into yeast vectors (AD/BD). Paired AD and BD constructs weretransformed into the yeast strain Y2HGold. Co-transformants were platedon both synthetic double dropouts medium (DDO) and triple dropoutsmedium (TDO) supplemented with X-a-Gal and Aureobasidin A. Interactionwas considered relevant when diploid yeasts were able to grow on bothDDO and TDO plates with blue color, while corresponding controls (AD orBD empty vectors) could not. Rcg1b ID showed direct binding with twocandidate effectors (ANTROT_70 and ANTROT_94).

To validate the two candidates in planta, the coding sequences of Rcg1,Rcg1b, ANTROT_70 and ANTROT_94 were transferred into a binary vector andintroduced into A. tumefaciens for co-infiltration assay in N.benthamiana. Co-expression of Rcg1b with ANTROT_70 was able to inducethe cell death phenotype in 30-50% infiltrated tobacco leaves, whileRcg1 was unable to recognize ANTROT_70 to cause cell death. However,co-expression of Rcg1b, ANTROT_70 and Rcg1 enhances the ability of Rcg1Bto recognize ANTROT_070 in N. benthamiana, with 60-70% of theinfiltrated leaves showing the cell death phenotype. Co-IP assay revealsthat Rcg1b physically interacts with both Rcg1 and ANTROT_70 while Rcg1does not interact with ANTROT_70. These results suggest Rcg1b andANTROT_70 directly interact with each other, while Rcg1 enhances theimmune responses elicited by the Rcg1b/ANTROT_70 interaction.

TABLE 6 ANTROT Effector Sequences SEQ ID NO: Sequence Description 1629ANTROT_70_GENOMIC 1630 ANTROT_70 CDS 1631 ANTROT_70 Protein

1. A method of validating a causal disease resistance gene comprising:a. Identifying at least one potential gene in a disease resistance locusin a disease resistance plant; b. Transfecting at least one allele of aplant pathogen effector gene and a luciferase gene into a maizeprotoplast, wherein the maize protoplast is derived from the diseaseresistance plant, and wherein the pathogen effector gene encodes apolypeptide comprising an amino acid sequence of at least 95% sequenceidentity, when compared to any one of SEQ ID NOs: 12, 2, 4, 10, 11,13-23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461,463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489,491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545,547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573,575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601,603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629,631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657,659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685,687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713,715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741,743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769,771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797,799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825,827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853,855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881,883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909,911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937,939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965,967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993,995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017,1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041,1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065,1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089,1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113,1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137,1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161,1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185,1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209,1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233,1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257,1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281,1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305,1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329,1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353,1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377,1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401,1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425,1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449,1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473,1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497,1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521,1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545,1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569,1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593,1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617,1619, 1626-1628, or 1631; c. Measuring luciferase activity; and d.Selecting a gene that produces a hypersensitive response in the presenceof the plant pathogen effector.
 2. The method of claim 1, wherein theplant pathogen effector gene has been validated as a plant pathogeneffector for the disease correlated with the disease resistance loci. 3.The method of claim 1, wherein the disease resistant donor plant is amaize plant.
 4. The method of claim 1, wherein the disease resistantdonor plant is a soybean plant.
 5. A method of selecting a diseaseresistant donor plant comprising: a. Transfecting at least one allele ofa plant pathogen effector gene comprising and a luciferase gene into amaize protoplast, wherein the maize protoplast is derived from a plantresistant to the plant pathogen, and wherein the pathogen effector geneencodes a polypeptide comprising an amino acid sequence of at least 95%sequence identity, when compared to any one of SEQ ID NOs: 12, 2, 4, 10,11, 13-23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 289, 291,293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543,545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571,573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599,601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711,713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739,741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795,797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823,825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851,853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879,881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907,909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935,937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963,965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991,993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015,1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039,1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063,1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087,1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111,1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135,1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159,1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183,1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207,1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231,1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255,1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279,1281, 1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303,1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327,1329, 1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351,1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375,1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399,1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423,1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447,1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471,1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495,1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519,1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543,1545, 1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567,1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591,1593, 1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615,1617, 1619, 1626-1628, or 1631; b. Measuring luciferase activity; and c.Selecting a maize plant that produces a hypersensitive response in thepresence of the plant pathogen effector.
 6. The method of claim 5,wherein the plant pathogen effector gene has been validated as a plantpathogen effector for the disease correlated with the disease resistanceloci.
 7. The method of claim 5, wherein the disease resistant donorplant is a maize plant.
 8. The method of claim 5, wherein the diseaseresistant donor plant is a soybean plant.
 9. (canceled)
 10. The methodof claim 5, further comprising crossing the selected maize plant with asecond maize plant.
 11. A protoplast comprising a predicted pathogeneffector gene, a luciferase gene, and a potential maize diseaseresistance gene, wherein the predicted pathogen effector gene ispredicted from a computational analysis, and wherein the pathogeneffector gene encodes a polypeptide comprising an amino acid sequence ofat least 95% sequence identity, when compared to any one of SEQ ID NOs:12, 2, 4, 10, 11, 13-23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315,317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343,345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371,373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399,401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427,429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455,457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483,485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511,513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539,541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567,569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595,597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623,625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651,653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679,681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707,709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735,737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763,765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791,793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819,821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847,849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875,877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903,905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931,933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959,961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987,989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013,1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037,1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061,1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085,1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109,1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133,1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157,1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181,1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205,1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229,1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253,1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277,1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301,1303, 1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325,1327, 1329, 1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349,1351, 1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373,1375, 1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397,1399, 1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421,1423, 1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445,1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469,1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493,1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517,1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541,1543, 1545, 1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565,1567, 1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589,1591, 1593, 1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613,1615, 1617, 1619, 1626-1628, or 1631; wherein the protoplast comprises amaize protoplast or a soybean protoplast.
 12. The protoplast of claim11, wherein the predicted pathogen effector gene and the luciferasereporter gene are both expressed from a single expression vector. 13-14.(canceled)
 15. A plant comprising a dsRNA targeting a pathogen effectorprotein, wherein the pathogen effector protein was identified orvalidated through a luciferase reporter protoplast assay, and whereinthe pathogen effector gene encodes a polypeptide comprising an aminoacid sequence of at least 95% sequence identity, when compared to anyone of SEQ ID NOs: 12, 2, 4, 10, 11, 13-23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391,393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419,421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447,449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475,477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503,505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531,533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559,561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587,589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615,617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643,645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727,729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755,757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783,785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839,841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867,869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895,897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923,925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951,953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979,981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005,1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029,1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053,1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077,1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101,1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125,1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149,1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173,1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197,1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221,1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245,1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269,1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293,1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315, 1317,1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339, 1341,1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363, 1365,1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387, 1389,1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411, 1413,1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435, 1437,1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461,1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485,1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509,1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533,1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555, 1557,1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579, 1581,1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603, 1605,1607, 1609, 1611, 1613, 1615, 1617, 1619, 1626-1628, or
 1631. 16-20.(canceled)
 21. A method to deploy a disease resistant plane, comprisingsurveying an effector sequence from a plant pest population to identifyallele diversity and allele frequency of the effector in a field pestpopulation.
 22. The method of claim 21, further comprising deploying aplant comprising an R-gene that interacts with the effector based on theallele specific data.
 23. The method of claim 21, wherein the effectorsequence has at least 95% sequence identity any one of SEQ ID NO:1626-1628, or
 1631. 24-57. (canceled)